Pharmacological vitreolysis

ABSTRACT

A method of treating or preventing a disorder, or a complication of a disorder, of an eye of a subject comprising contacting a vitreous and/or aqueous humor with a composition comprising a truncated form of plasmin comprising a catalytic domain of plasmin (TPCD). TPCDs include, but are not limited to, miniplasmin, microplasmin and derivatives and variants thereof. The methods of the invention can be used to reduce the viscosity of the vitreous, liquefy the vitreous, induce posterior vitreous detachment, reduce hemorrhagic blood from the eye, clear or reduce materials toxic to the eye, clear or reduce intraocular foreign substances from the eye, increase diffusion of a composition administered to an eye, reduce extraretinal neovascularization and any combinations thereof. The method can be used in the absence of, or as an adjunct to, vitrectomy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/156,907, filed Jun. 5, 2008, entitled “Pharmacological Vitreolysis”,which is a continuation of U.S. application Ser. No. 10/729,475,entitled, “Pharmacological Vitreolysis,” filed Dec. 5, 2003, which inturn claims the benefit of priority to Great Britain Application No.0228409.9 filed Dec. 6, 2002, the contents of both of which are hereinincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates generally to methods of treating orpreventing a disorder, or a complication of a disorder, of the mammalianeye. More specifically, the present invention relates to the use of atruncated plasmin protein comprising a catalytic domain in methods oftreating or preventing a disorder, or a complication of a disorder, ofthe mammalian eye.

BACKGROUND OF THE INVENTION

The adult human eye is a slightly asymmetrical sphere with anapproximate sagittal diameter of 24 to 25 mm, a transverse diameter of24 mm, and a volume of about 6.5 cc. The human eye can be divided intothree different layers namely, an external layer, an intermediate layerand an internal layer. The external layer of the eye consists of thesclera, which is often referred to as the “white of the eye,” and thecornea, which covers the front of the eye. The intermediate layer isdivided into an anterior portion and a posterior portion; the anteriorportion consists of the circular pigmented iris, the crystalline lensand ciliary body, while the posterior portion consists of the choroidlayer. The internal layer consists of the retina, which is the sensorypart of the eye. The retina is essentially a layer of nervous tissue,which runs along the inside rear surface of the choroid layer and can bedivided into an optic portion and a non-optic portion. The opticportion, which participates in the visual mechanism, contains the rodsand cones that are the effectual organs of vision.

The human eye can also be divided into three chambers. The anteriorchamber between the cornea and the iris, and the posterior chamberbetween the iris and the crystalline lens, are filled with aqueoushumor. In contrast, the vitreous chamber between the crystalline lensand the retina is filled with a more viscous liquid, called the vitreous(also known as the vitreous body or vitreous humor). The vitreous humorin a normal eye is a clear gel occupying about 80% of the volume of theeyeball. Light that enters the eye through the cornea, pupil, and lens,is transmitted through the vitreous to the retina.

The vitreous humor of a normal human eye is a gel that is roughly 99%water and 1% macromolecules. These macromolecules include a network ofcollagen fibrils, hyaluronic acid, soluble glycoproteins, sugars andother low molecular weight metabolites. Type II collagen is theprincipal fibrillar collagen of the vitreous, but the vitreous alsocontains collagen types V, IX, and XI. The posterior portion of thevitreous body, the posterior hyaloid surface (also known as theposterior vitreous cortex), is in direct contact with the inner retinalsurface most prominently at the vitreous base, optic disc, and along themajor retinal vessels. Normal adhesion of the vitreous to the retina ismediated by cellular and molecular interactions between the posteriorvitreous cortex and the inner limiting membrane (ILM) of the retina. TheILM is essentially the basement membrane of retinal Mueller cells. TheILM contains collagen types I and IV, glycoproteins such as laminin andfibronectin and other glycoconjugates. These components are thought tobridge and bind collagen fibers between the vitreous and the ILM.

With age, the vitreous humor changes from gel to liquid and as it doesso it gradually shrinks and separates from the ILM of the retina. Thisprocess is known as “posterior vitreous detachment” (PVD) and is anormal occurrence after age 40. However, degenerative changes in thevitreous may also be induced by pathological conditions such asdiabetes, Eale's disease and uveitis. Also, PVD may occur earlier thannormal in nearsighted people and in those who have had cataract surgery.Usually, the vitreous makes a clean break from the retina. Occasionally,however, the vitreous adheres tightly to the retina in certain places.These small foci of resisting, abnormally firm attachments of thevitreous can transmit great tractional forces from the vitreous to theretina at the attachment site. This persistent tugging by the vitreousoften results in horseshoe-shaped tears in the retina. Unless theretinal tears are repaired, vitreous fluid can seep through this tearinto or underneath the retina and cause a retinal detachment, a veryserious, sight-threatening condition. In addition, persistent attachmentbetween the vitreous and the ILM can result in bleeding from rupture ofblood vessels, which results in the clouding and opacification of thevitreous.

The development of an incomplete PVD has an impact on many vitreoretinaldiseases including vitreomacular traction syndrome, vitreous hemorrhage,macular holes, macular edema, diabetic retinopathy, diabetic maculopathyand retinal detachment. Thus, an important goal of vitreous surgery isto separate the vitreous from the retina in a manner that preventsvitreous traction.

In order to remove the vitreous from the eye, a microsurgical procedurecalled vitrectomy is usually performed. In this procedure the vitreousis removed from the eye with a miniature handheld cutting device whilesimultaneously replacing the removed vitreous with saline solution toprevent collapse of the eye. Surgical removal of the vitreous using thismethod is highly skill-dependent, and complete removal of the corticalvitreous remains a difficult task. Furthermore, mechanical vitrectomycarries the risk of complications such as scarring, tearing and otherdamage to the retina. Obviously, such damage is highly undesirable as itcan compromise the patient's vision after surgery.

Thus, alternative methods to remove the vitreous from the retina havebeen the focus of recent investigation. Such methods have explored theuse of enzymes and chemical substances, which can be used toinduce/promote liquefaction of the vitreous and/or separation of thevitreoretinal interface (PVD). These approaches, which are referred toas “pharmacological vitrectomy,” have included several proteolyticenzymes such as alpha-chymotrypsin, hyaluronidase, bacterialcollagenase, chondroitinase and dispase, which have been injectedintravitreally in experimental and/or clinical trials to induce PVD.However, most of these techniques do not release the posterior hyaloidfrom the ILM completely or without complications. In addition, inseveral of these cases, the risk of adverse reactions is high. Forexample, the use of bacterial proteases in mammalian systems generatesan immune response, which leads to proliferative vitreoretinopathyresulting in complex retinal re-detachment. Collagenase has beenreported to liquefy the vitreous, but it has also been shown to disruptthe outer layers of the retina. Alpha-chymotrypsin has been reported toproduce peripapillary and vitreous hemorrhage in the injected eyes.Finally, dispase has been reported to cause toxicity to the inner layerof the retina 15 minutes after injection. Depending on the concentrationof dispase used, proliferative retinopathy or epiretinal cellularmembranes can develop in the injected eyes.

Given the immunogenicity and other adverse effects of bacterialproteases, pharmacological vitreolysis using endogenous human derivedproteases may be desirable. Plasmin is a serine protease derived fromplasminogen. Plasminogen is an important component of mammalian blood.Human plasminogen is a single chain glycoprotein consisting of 791 aminoacids, which has a molecular weight of about 92,000 daltons (seeForsgren M. et al., FEBS Lett. 213(2):254-60, 1987). Native plasminogenwith an amino-terminal glutamic acid (termed “Glu-plasminogen”) isconverted by limited digestion by plasmin of the Arg₆₈-Met₆₉,Lys₇₇-Lys₇₈, or Lys₇₈-Val₇₉ peptide bonds to proteins commonlydesignated as “Lys-plasminogen.” Activation of plasminogen byplasminogen activators such as urokinase or streptokinase, cleaves thepeptide bond between Arg₅₆₁ and Val₅₆₂ converting the plasminogenmolecule into a double chain, enzymatically active form called plasmin.Plasmin contains two polypeptides, a heavy A chain connected by twodisulphide bonds to a light B chain; the B chain contains the serineprotease catalytic domain. The serine protease catalytic activity ofplasmin has been implicated in its ability to dissolve blood clots invivo.

Recently, plasmin has also been suggested as an adjunct for vitrectomy.In addition, autologous plasmin enzyme (APE) has been suggested as anagent for pharmacological vitrectomy. However, there are severaldisadvantages associated with the use of plasmin. First, so far allclinical interventions with plasmin have relied on the use of APE, theisolation of which necessitates a laborious and time-consuming processinvolving drawing of a patient's blood, isolation of plasminogen,activation of the isolated plasminogen to plasmin, and purification andsterility testing of the plasmin enzyme. Furthermore, this procedure canbe costly and the presence of blood-borne pathogens can furthercomplicate this procedure. Also, plasmin is highly prone to degradationand thus cannot be stored for prolonged periods prior to its use. Afurther disadvantage is plasmin's large molecular weight, which rangesbetween 65,000 and 83,000 daltons. Thus, the diffusion of largemolecules like plasmin from its injected position in the vitreous to thevitreoretinal interface would be hindered compared to smaller molecules.

Accordingly, there is a need in the art for methods of treating orpreventing disorders, or complications of disorders, of the eye of asubject that overcome the disadvantages of plasmin, for pharmacologicalvitreolysis. Specifically, there is a need for methods of treating orpreventing a disorder, or a complication of a disorder, of the eye usingsmaller molecules than plasmin, which can diffuse through the vitreousto the vitreoretinal interface faster than plasmin, and which can bereadily obtained in large quantities without the delay and otherattendant problems of isolating autologous plasmin enzyme on apatient-by-patient basis.

SUMMARY OF THE INVENTION

The present invention provides methods of treating or preventing adisorder, or a complication of a disorder, of the eye of a subject usinga composition comprising a truncated plasmin protein comprising acatalytic domain of plasmin (TPCD). In one embodiment, a TPCD isselected from the group consisting of miniplasmin, recombinantminiplasmin, stabilized miniplasmin, stabilized, recombinantminiplasmin, variants of miniplasmin, microplasmin, recombinantmicroplasmin, stabilized microplasmin, stabilized, recombinantmicroplasmin, variants of microplasmin, and any combinations thereof.

The present invention also provides methods of treating or preventing adisorder, or a complication of a disorder, of the eye of a subject usinga composition comprising a modified TPCD. A modified TPCD is a TPCD,which comprises a modified catalytic domain of plasmin.

The present invention provides methods of treating or preventing adisorder, or a complication of a disorder, of the eye of a subject bycontacting a vitreous and/or an aqueous humor of the subject with acomposition comprising a TPCD. The present invention provides methods oftreating or preventing eye disorders such as, but not limited to,retinal detachment, retinal tear, vitreous hemorrhage, diabetic vitreoushemorrhage, proliferative diabetic retinopathy, non-proliferativediabetic retinopathy, age-related macular degeneration, macular holes,vitreomacular traction, macular pucker, macular exudates, cystoidmacular edema, fibrin deposition, retinal vein occlusion, retinal arteryocclusion, subretinal hemorrhage, amblyopia, endophthalmitis,retinopathy of prematurity, glaucoma, retinitis pigmentosa and anycombinations thereof. The methods of the invention can be practicedindependent of vitrectomy, or as an adjunct to vitrectomy.

The present invention also provides methods of treatment or preventionof an eye disorder, or a complication of an eye disorder, of a subjectcomprising administering to the subject a composition comprising atleast two TPCDs. In one embodiment of this aspect of the invention, acomposition is administered to a subject by contacting a vitreous and/oran aqueous humor with a composition comprising at least two TPCDs.

The present invention further encompasses methods of treatment orprevention of an eye disorder, or a complication of an eye disorder,comprising providing a subject with a first composition comprising atleast one TPCD, and a second composition comprising at least one TPCD.In one embodiment of this aspect of the invention, a first compositioncomprising at least one TPCD and a second composition comprising atleast one TPCD are provided to a subject by contacting a vitreous and/oran aqueous humor. In another embodiment of this aspect of the invention,the TPCDs of the first composition comprising at least one TPCD and thesecond composition comprising at least one TPCD are the same TPCD. Inyet another embodiment of this aspect of the invention, the TPCDs of thefirst composition comprising at least one TPCD and the secondcomposition comprising at least one TPCD are different TPCDs. In afurther embodiment of this aspect of the invention, the firstcomposition comprising at least one TPCD and the second compositioncomprising at least one TPCD are administered to a subject atsubstantially the same time. In yet another embodiment, the firstcomposition comprising at least one TPCD and the second compositionscomposition comprising at least one TPCD are administered to a subjectat separate times.

The present invention additionally provides methods of treatment orprevention of an eye disorder, or a complication of an eye disorder, ofa subject by administering a composition comprising at least one TPCDand at least one second agent to the subject. A second agent includesany substance that is useful either alone, or in combination with aTPCD, in treating or preventing an eye disorder or a complication of aneye disorder. A second agent, includes without limitation,hyaluronidase, dispase, chondroitinase, collagenase, RGD containingpeptides, anti-integrin antibody, urea, hydroxyurea, thiourea, P2Yreceptor agonists, and any angiogenic inhibitors including, but notlimited to, VEGF inhibitors and PlGF inhibitors.

The present invention also encompasses methods of treatment orprevention of an eye disorder, or a complication of an eye disorder, ofa subject by administering to the subject a composition comprising atleast one TPCD prior to or after administration of a compositioncomprising a second agent.

The methods of the invention can be used to treat or prevent an eyedisorder, or a complication of an eye disorder, of a subject byeffecting one or more outcomes including, but not limited to, reducingthe viscosity of the vitreous, liquefying the vitreous, inducingposterior vitreous detachment, clearing or reducing hemorrhagic bloodfrom the vitreous and/or aqueous humor, clearing or reducing intraocularforeign substances from the vitreous and/or aqueous humor, clearing orreducing materials toxic to the retina, increasing diffusion of an agentor a composition administered to the vitreous and/or aqueous humor,reducing extraretinal neovascularization and any combinations thereof.

The present invention also provides methods of performing a vitrectomycomprising contacting the vitreous of a subject with a compositioncomprising a TPCD. The contacting step can be performed prior to or atthe same time as the vitrectomy or independent of vitrectomy.

The present invention also provides a composition comprising at leasttwo TPCDs.

The present invention further provides a composition comprising at leastone TPCD and at least one second agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the DNA (SEQ ID NO:9) and amino acid sequence (SEQ IDNO:10) of human plasminogen.

FIG. 2 provides the DNA (SEQ ID NO:3) and amino acid sequence (SEQ IDNO:4) of human microplasminogen.

FIG. 3 provides the DNA (SEQ ID NO:7) and amino acid sequence (SEQ IDNO:8) of human miniplasminogen.

FIG. 4 shows the effect of treating porcine eyes with microplasmin.Panel A is a low magnification image (11×) of the mid-peripheral retinaafter slow dehydration of a porcine eye treated with 0.125 mg ofmicroplasmin in 0.1 ml BSS PLUS® for 120 minutes. In the centre of thisimage, is a vitreous strand. It is likely that this vitreous strand isvitreous that has collapsed onto the retinal surface. Most of theretinal surface is free of vitreous as shown in the remaining panels.Panel B shows an area of bare retina adjacent to a blood vessel(magnification 800×). Few cells are seen on the retinal surface. Theirregular surface is that of the vessel itself Panel C and D aremagnifications of the retinal area in B at 1200× and 3600× magnificationrespectively showing a smooth retinal surface largely devoid of vitreousor cellular material. At 3600× only a few fibrillar strands are visible.Panel E is an image at a magnification of 1500× essentially showing thesame findings as in Panel C at a more central retinal location, whilepanel F shows the coarse granular structure of the vitreous, which haslost its fibrillar structure. The structure of the vitreous in themicroplasmin treated eyes is very different in appearance compared tothe vitreous in control eyes (data not shown).

FIG. 5 shows that ciliary processes in the porcine eye are intact after120 minute treatment with microplasmin (Panels A and B).

FIG. 6 provides scanning electron micrographs (magnification of 3600×)of the vitreoretinal interface in human post-mortem eyes. Intravitrealinjection of 62.5 μg of microplasmin resulted in posterior vitreousdetachment (PVD) leaving discontinuous remnants of collagen fibrilscovering the ILM (Panel A). 125 μg (Panel B) and 188 μg (Panel C) ofmicroplasmin produced complete PVD and a bare ILM. Panel D shows thecompression of collagen fibrils towards the ILM in an eye treated with62.5 μg microplasmin and gas. Panel E shows complete PVD followingtreatment with 125 μg microplasmin and gas. Unlike the PVD observed inmicroplasmin treated eyes, there is a dense network of collagen fibrilsin the control eye (Panel F).

FIG. 7 provides transmission electron micrographs of the ILM in humanpost-mortem eyes. Note the absence of collagen fibrils (arrows) on theILM in the microplasmin-treated eye (Panel A, magnification 13,600×). Incontrast, collagen fibrils are still present (arrows) on the ILM in thecontrol eye (Panel B; magnification 6800×).

FIG. 8 presents scanning electron micrographs (magnification 3600×) ofthe vitreoretinal interface in cat eyes. Intravitreal injection of 25 μgof microplasmin left remnants of collagen fibrils on the ILM one dayafter treatment (Panel A). Three days after treatment, 25 μg ofmicroplasmin resulted in complete PVD (Panel B). Remnants of collagenfibrils were observed three days following treatment with 14.5 μg ofmicroplasmin (Panel C). A bare ILM was observed three weeks afterinjection of 14.5 μg of microplasmin (Panel D) and 25 μg of microplasmin(Panel E). In striking contrast, the control eye showed a dense attachedcortical vitreous (Panel F).

FIG. 9 presents the results of light microscopy studies of semi-thinsections of cat eyes. These studies showed that the normalcytoarchitecture of the retina observed in control eyes (Panel B) wasalso observed in microplasmin-treated eyes (Panel A). Transmissionelectron microscopy revealed a well-preserved inner retina and ILM inmicroplasmin-treated eyes (Panels C and E) as observed in control eyes(Panels D and F). The magnification used for Panels A and B was 250×;the magnification for Panels C and D was 6000×; while the magnificationfor Panels E and F was 30,000×.

FIG. 10 presents the results of confocal laser scanning microscopy withprobes to glial fibrillic acidic protein (Panels A and B, green) andvimentin (Panels C and D, red). There is no difference in the stainingof GFAP and vimentin between microplasmin-treated eyes (Panels A and C)and control eyes (Panels B and D). Double-label immunohistochemistrywith probes to synaptophysin (green) and neurofilament (red) also showsno difference between microplasmin-treated eyes (Panel E) and a controleyes (Panel F). Magnification for Panels A, B and C was 400×;magnification for Panel D was 250×; and magnification for Panels E and Fwas 160×.

FIG. 11 presents a time correlation function (TCF) of whole porcinevitreous as compared to a solution of 20 nm polystyrene nanospheres. Inthe vitreous there are two-components to the curve. The early (fast)component is due to the presence of hyaluronan (HA) that is freelydiffusible and exhibits considerable molecular (Brownian) motion. Thelate (slow) component is due to collagen, which is larger and diffusesless freely (stiffer), resulting in slower Brownian movement. Incontrast, the solution of polystyrene nanospheres has only one component(monodisperse) that is very fast because of the small size of thenanospheres and their perfectly spherical structure allowing for veryrapid movements in the solution.

FIG. 12 presents normalized time correlation functions for 5 porcineeyes undergoing microplasmin (μPli) pharmacologic vitreolysis atdifferent doses and a solution of 20 nm polystyrene nanospheres. DLSmeasurements were made in the optical axis, 4 mm below the air/vitreousinterface. In the untreated (vehicle) vitreous there are two-componentsto the curve. The early (fast) component is due to the presence ofhyaluronan (HA) that is freely diffusible and exhibits considerablemolecular (Brownian) motion. The late (slow) component is due tocollagen, which is larger and diffuses less freely (stiffer), resultingin slower Brownian movement. At the other extreme, the solution ofpolystyrene nanospheres has only one component (monodisperse) that isvery fast because of the small size of the nanospheres and theirperfectly spherical structure allowing for very rapid movements in thesolution. With increasing doses of μPli there is a decrease in the slopeof the TCF with disappearance of the slow component (larger molecularspecies) ultimately approaching the TCF of pure 20 nm nanospheres, i.e.,all smaller size molecular species.

FIG. 13 presents representative photographs of porcine eyes afterinjection of microplasmin and fluorescein. Both images are of the sameeye, with the right image captured 20 minutes after the left image,demonstrating fluorescein diffusion in the vitreous.

FIG. 14 presents representative photographs of porcine eyes afterinjection of plasmin and fluorescein. Both images are of the same eye,with the bottom image captured 20 minutes after the top image,demonstrating fluorescein diffusion in the vitreous to a lesser degreethan that seen with microplasmin-treated eyes (FIG. 13).

DETAILED DESCRIPTION OF THE INVENTION

The patent applications, patents, and literature references cited hereinindicate the knowledge of those of ordinary skill in this field and arehereby incorporated by reference in their entirety. In the case ofinconsistencies between any reference cited herein and the specificteachings of the present disclosure, this disclosure will prevail.

The following detailed description and the accompanying examples areprovided for purposes of describing and explaining only certainpreferred embodiments of the invention, and are not intended to limitthe scope of the invention in any way. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art to which thisinvention belongs.

Prior to setting forth the invention in detail, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereafter.

DEFINITIONS

“treating,” means the reduction or amelioration of any medical disorderto any extent, and includes, but does not require, a complete cure ofthe disorder.“preventing” means to defend or protect against the development of adisorder, i.e., to function as a prophylactic.“disorder” means any disease, dysfunction, syndrome, condition, pain, orany combination thereof. Disorder also includes any complications fromany disease, dysfunction, syndrome, condition, pain or any combinationthereof“subject” means any mammal, particularly a human.“contacting” means any mode of administration that results ininteraction between a composition and an object being contacted (e.g.,vitreous, aqueous humor, etc.). The interaction of the composition withthe object being contacted can occur at substantially the same time asthe administration of the composition, over an extended period of timestarting from around the time of administration of the composition, orbe delayed from the time of administration of the composition.“composition” means a combination or mixture of one or more substances.“substance” means that which has mass and occupies space.“foreign substance” means any substance that is determined by a medicaldoctor, clinician, veterinarian or researcher to be harmful or toxic tothe eye of a subject and/or to be a substance that is not normally foundin a healthy mammalian eye.“ophthalmologically acceptable carrier” is a substance with which asecond substance (for e.g., a TPCD) can be combined, without making thesecond substance unsuitable (as determined by a medical doctor,clinician, veterinarian or researcher) for its intended use in the eyeof a subject. Non-limiting examples of an ophthalmologically acceptablecarrier include balanced salt solution (BSS) and BSS-PLUS®.“pharmaceutically acceptable carrier” includes, without limitation,water, buffered saline, polyol (for e.g., glycerol, propylene glycol,liquid polyethylene glycol), or suitable mixtures thereof. Otherexamples of pharmaceutically acceptable carriers and methods for makingsuch carriers and formulations thereof are found, for example, inRemington's Pharmaceutical Sciences (20th Edition, A. Gennaro (ed.),Lippincott, Williams & Wilkins, 2000).“an effective amount” means an amount of a substance or composition thatelicits a response in an eye of a human or other mammal that is beingsought by a researcher, veterinarian, medical doctor or other clinician.“inducing” means to bring about or stimulate the occurrence of a desiredresult.“reduce” means to decrease to any extent.“toxic effects to the eye” means any adverse effect to the eye of asubject that is determined to be harmful to the subject by a researcher,veterinarian, medical doctor or other clinician.“vitreous” means the vitreous humor, also referred to as the vitreousbody, which occupies the chamber between the crystalline lens of the eyeand the retina.“TPCD” is an acronym for “truncated plasmin protein comprising acatalytic domain of plasmin.” A “truncated plasmin protein” means anyplasmin protein obtained by deleting one or more amino acids ofVal₇₉-plasmin (i.e., amino acids 79-791 of human plasminogen), whereinthe resulting protein possesses serine protease catalytic activity. Suchamino acid deletions can be at the N-terminus (resulting in TPCDsconsisting for example of, amino acids 444-791, 543-791, or 562-791 ofSEQ ID NO:10) and/or at the C-terminus and/or at any internal positionor positions of amino acids 79-791 of SEQ ID NO:10. It is to beunderstood that if a truncated protein derived from SEQ ID NO:10 is madeas an enzymatically inactive form, it must be activated using aplasminogen activator to convert it into the corresponding active formof the truncated protein. For example, if a protein consisting of aminoacids 543-791 of SEQ ID NO:10 is made recombinantly, it is highly likelythat the protein will not be in its enzymatically active form. Thus, theprotein should be treated with a plasminogen activator to cleave thepeptide bond between Arg₅₆₁ and Val₅₆₂, thereby activating the protein.Non-limiting examples of a TPCD include miniplasmin, recombinantminiplasmin, stabilized miniplasmin, stabilized, recombinantminiplasmin, variants of miniplasmin, microplasmin, recombinantmicroplasmin, stabilized microplasmin, stabilized, recombinantmicroplasmin and variants of microplasmin wherein, the variants ofmicroplasmin and miniplasmin include a catalytic domain of plasmin.“plasmin protein” means any protein made or derived from the amino acidsequence of human plasminogen (SEQ ID NO:10) that has a cleavage of thepeptide bond between Arg₅₆₁ and Val₅₆₂ of human plasminogen. Thecleavage of the peptide bond between Arg₅₆₁ and Val₅₆₂ can beaccomplished using plasminogen activators. Non-limiting examples ofplasmin proteins include Lys-plasmin, miniplasmin and microplasmin.“catalytic domain of plasmin” means an amino acid sequence of about130-240 amino acids derived from amino acids 543 to 791 of SEQ ID NO:10(human plasminogen), which includes the catalytic triad of plasminnamely, His₆₀₃, Asp₆₄₆ and Ser₇₄₁, wherein the amino acid sequencepossesses serine protease activity.“modified catalytic domain of plasmin” means a catalytic domain ofplasmin that has been altered by changing the amino acid sequence of thecatalytic domain by addition and/or deletion and/or substitution of oneor more amino acids. Of course it is to be understood that the aminoacids corresponding to the catalytic triad of plasmin namely, His₆₀₃,Asp₆₄₆ and Ser₇₄₁, are not altered. The modification may increase,decrease or leave unchanged the plasmin-like catalytic activity of theprotein. For example, the modified catalytic domain of microplasmin andminiplasmin may increase, decrease or leave unchanged the catalyticactivity of these proteins.“modified TPCD” is a TPCD containing a modified catalytic domain ofplasmin, wherein the TPCD possesses plasmin-like serine proteasecatalytic activity.“second agent” means any substance that can be used, either by itself,or in combination with a TPCD, in treating or preventing an eye disorderor a complication of an eye disorder of a subject. Preferably the secondagent does not prevent the catalytic activity of a TPCD.“stabilizing a protein” means protecting a protein from degradationand/or inactivation through the use of one or more stabilizing agents.

Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice or testing of the presentinvention, the preferred methods and materials are described below.

Pharmacological vitreolysis is a method of using one or moreproteinaceous and/or chemical and/or nucleic acid agents to treat orprevent a disorder, or a complication of a disorder, of an eye of asubject. The present invention provides methods of pharmacologicalvitreolysis using at least one truncated plasmin protein comprising acatalytic domain (TPCD). Specifically, the present invention providesmethods of treatment or prevention of eye disorders, or complications ofeye disorders, by contacting the vitreous and/or aqueous humor with aneffective amount of a composition comprising a TPCD. These methodsresults in outcomes such as, but not limited to, liquefaction of thevitreous, posterior vitreous detachment, reduction or clearing ofhemorrhagic blood from the vitreous and/or aqueous humor, reduction orclearing of intraocular foreign substances from the vitreous and/oraqueous humor, increasing diffusion of an agent or compositionadministered to the vitreous and/or aqueous humor, decreasingextraretinal neovascularization, and any combinations thereof. Thesemethods may be used either as an adjunct to vitrectomy, or in theabsence of vitrectomy.

Accordingly, the present invention provides, as a first aspect, a methodof treating or preventing a disorder, or a complication of a disorder,of the eye of a subject comprising contacting the vitreous and/oraqueous humor with an effective amount of a composition comprising aTPCD. In one embodiment, a TPCD has a molecular weight less than about40,000 daltons. In another embodiment, a TPCD has a molecular weight ofabout 26,500 daltons in reduced form or about 29,000 daltons innon-reduced form. In yet another embodiment a TPCD has a molecularweight of between about 20,000 and 30,000 daltons. In a furtherembodiment, a TPCD has a molecular weight of less than about 20,000daltons.

In a second aspect, the present invention provides a method of treatingor preventing a disorder, or a complication of a disorder, of the eye ofa subject comprising contacting the vitreous and/or aqueous humor withan effective amount of a composition comprising at least two TPCDs.

In a third aspect, the present invention provides a method of treatingor preventing a disorder, or a complication of a disorder, of the eye ofa subject comprising contacting the vitreous and/or aqueous humor withan effective amount of a first composition comprising at least one TPCDand an effective amount of a second composition comprising at least oneTPCD. In one embodiment of this aspect of the invention, the firstcomposition comprising at least one TPCD and the second compositioncomprising at least one TPCD can comprise the same TPCD In anotherembodiment of this aspect of the invention, the first compositioncomprising at least one TPCD and the second composition comprising atleast one TPCD can comprise different TPCDs. In a further embodiment ofthis aspect of the invention, the first and second compositions may beadministered to a subject at substantially the same time or at differenttimes.

In a fourth aspect, the present invention provides a method of treatingor preventing a disorder, or a complication of a disorder, of the eye ofa subject comprising contacting the vitreous and/or aqueous humor withan effective amount of a composition comprising at least one TPCD and atleast one second agent. In this aspect of the invention, the secondagent is not intended to be a TPCD.

In a fifth aspect, the present invention provides a method of treatingor preventing a disorder, or a complication of a disorder, of the eye ofa subject comprising contacting the vitreous and/or aqueous humor withan effective amount of a composition comprising at least one TPCD priorto, at the same time as, or after contacting the vitreous and/or aqueoushumor with an effective amount of a composition comprising at least onesecond agent.

In a sixth aspect, the present invention provides a method of liquefyingthe vitreous comprising contacting the vitreous and/or aqueous humorwith an effective amount of a composition comprising at least one TPCD.In one embodiment of this aspect of the invention, the liquefaction ofthe vitreous decreases the viscosity of the vitreous humor. In otherembodiments of the invention, the liquefaction of the vitreous increasesthe rate of clearance from the vitreous cavity and/or aqueous humor ofblood, deposited material, foreign substances and/or materials toxic tothe eye, especially the retina. In another embodiment of this aspect ofthe invention, the liquefaction of the vitreous decreases extraretinalneovascularization. In yet another embodiment of this aspect of theinvention, the liquefaction of the vitreous increases the diffusion ofan agent or composition administered to the vitreous and/or aqueoushumor. In a further embodiment of this aspect of the invention, theliquefaction of the vitreous helps in the removal of the vitreous duringstandard vitrectomy or 25 Gauge (or smaller) vitrectomy.

In a seventh aspect, the present invention provides a method of inducingposterior vitreous detachment comprising contacting the vitreous and/oraqueous humor with an effective amount of a composition comprising atleast one TPCD.

In any of the first to seventh aspects of the invention described above,the step of contacting the vitreous and/or aqueous humor with acomposition comprising a TPCD can be performed as an adjunct to, or inthe absence of vitrectomy.

In an eighth aspect, the present invention provides a method ofperforming a vitrectomy comprising the step of contacting the vitreousand/or aqueous humor with a composition comprising at least one TPCD.The contacting step can be performed at the same time as, or prior tovitrectomy.

In a ninth aspect, the present invention provides a compositioncomprising at least two TPCDs.

In a tenth aspect, the present invention provides a compositioncomprising at least one TPCD and at least one second agent.

In one embodiment of all aspects of the present invention, a TPCD isselected from the group consisting of miniplasmin, recombinantminiplasmin, stabilized miniplasmin, stabilized, recombinantminiplasmin, variants of miniplasmin, microplasmin, recombinantmicroplasmin, stabilized microplasmin, stabilized, recombinantmicroplasmin, variants of microplasmin, and any combinations thereof. Inanother embodiment of all aspects of the invention, the methods oftreatment or prevention of an eye disorder, or complications of an eyedisorder, and methods of performing a vitrectomy result in theamelioration of an eye disorder by one or more of the followingoutcomes: reducing the viscosity of the vitreous, liquefying thevitreous, inducing posterior vitreous detachment, clearing or reducinghemorrhagic blood from the vitreous, vitreous cavity and/or aqueoushumor, clearing or reducing intraocular foreign substances from thevitreous, vitreous cavity and/or aqueous humor, clearing or reducingmaterials toxic to the retina from the vitreous, vitreous cavity and/oraqueous humor, increasing the diffusion of an agent or a compositionadministered to the vitreous and/or aqueous humor, or reducing retinalneovascularization. In yet another embodiment of all aspects of theinvention, the eye disorder or complication of an eye disorder sought tobe treated or prevented is selected from the group consisting of retinaldetachment, retinal tear, vitreous hemorrhage, diabetic vitreoushemorrhage, proliferative diabetic retinopathy, non-proliferativediabetic retinopathy, age-related macular degeneration, macular holes,vitreomacular traction, macular pucker, macular exudates, cystoidmacular edema, fibrin deposition, retinal vein occlusion, retinal arteryocclusion, subretinal hemorrhage, amblyopia, endophthalmitis,retinopathy of prematurity, glaucoma, retinitis pigmentosa, and anycombination thereof.

A TPCD includes any truncated plasmin protein comprising a catalyticdomain of plasmin. A truncated plasmin protein encompasses any plasminprotein obtained by deleting one or more amino acids of Val₇₉-plasmin(i.e., amino acids 79-791 of human plasminogen), wherein the resultingprotein possesses serine protease catalytic activity. Thus, all TPCDsmust contain the catalytic triad of the plasmin serine protease domainnamely, His₆₀₃, Asp₆₄₆ and Ser₇₄₁. Truncations of Val₇₉-plasmin can bemade by deletions of one or more amino acids in amino acids 79-791 ofSEQ ID NO:10 (human plasminogen). Such deletions can be at theN-terminus, C-terminus or at an internal location of amino acids 79-791of SEQ ID NO:10. It should be understood that if a protein resultingfrom a truncation of amino acids 79-791 of SEQ ID NO:10 (humanplasminogen) is made in an enzymatically inactive form, the protein mustbe converted to its active form using a plasminogen activator, to beconsidered a TPCD. Plasminogen activators cleave the peptide bondbetween Arg₅₆₁ and Val₅₆₂, thereby activating a protein. In oneembodiment a TPCD includes proteins consisting essentially of aminoacids 444-791 of SEQ ID NO:10. In another embodiment, a TPCD includesproteins with one or more amino acid deletions in amino acids 444-791 ofhuman plasminogen, wherein the resulting protein possesses serineprotease catalytic activity. In another embodiment, a TPCD includesproteins consisting essentially of amino acids 543-791 of SEQ ID NO:10.In yet another embodiment a TPCD includes proteins with one or moredeletions in amino acids 543-791 of human plasminogen, wherein theresulting protein possesses serine protease catalytic activity. Inanother embodiment, a TPCD includes proteins consisting essentially ofamino acids 562-791 of SEQ ID NO:10. In another embodiment a TPCDincludes proteins with one or more deletions in amino acids 562-791 ofhuman plasminogen, wherein the resulting protein possesses serineprotease catalytic activity. The deletions can be at the N-terminus,C-terminus or at an internal location of amino acids 444-791, 543-791and 562-791 of SEQ ID NO:10 (human plasminogen), respectively. Methodsof making amino acid deletions in a protein are well known to those ofordinary skill in the art (for e.g., Current Protocols in MolecularBiology, Ausubel et al. (eds.), John Wiley & Sons, 2001; and MolecularCloning: A Laboratory Manual, Third Edition, Sambrook and Russell,2000). The catalytic activity of a TPCD can be determined by measuringthe amidolytic activity of the TPCD, using the chromogenic substrateS2403 (Chromogenix, Antwerp, Belgium) (see Example 2), any otherchromogenic substrate, or by any other methods known in the art.

The present invention also envisions the use of a modified TPCD. Amodified TPCD is a TPCD with a modified form of the catalytic domain ofplasmin, wherein the modified TPCD possesses plasmin-like serineprotease catalytic activity. Modifications to the catalytic domaininclude amino acid insertions and/or deletions and/or substitutions inthe catalytic domain of plasmin. However, the amino acids correspondingto the catalytic triad of the plasmin serine protease domain namely,Histidine₆₀₃, Aspartic Acid₆₄₆ and Serine₇₄₁ are not altered. Preferablythe modifications of the catalytic domain involve one or moreconservative substitution(s) of amino acids that are not part of thecatalytic triad. Conservative amino acid substitutions and methods ofmaking such conservative amino acid substitutions are well known to oneof ordinary skill in the art (see, e.g., Current Protocols in MolecularBiology, supra; and Molecular Cloning: A Laboratory Manual, supra).These modifications may increase, decrease or leave unchanged thecatalytic activity of the original domain. The catalytic activity of amodified TPCD can be determined by measuring the amidolytic activity ofthe TPCD, using the chromogenic substrate S2403 (Chromogenix, Antwerp,Belgium) (see Example 2) or by any other methods known in the art.

TPCD includes, but is not limited to, miniplasmin, recombinantminiplasmin, stabilized miniplasmin, stabilized, recombinantminiplasmin, variants of miniplasmin, microplasmin, recombinantmicroplasmin, stabilized microplasmin, stabilized, recombinantmicroplasmin, and variants of microplasmin. Variants of microplasmin andminiplasmin include shorter forms of microplasmin and miniplasmin thatcan be produced by amino acid deletions from these proteins. Allvariants of microplasmin and miniplasmin are expected to have serineprotease catalytic activity, even if they do not possess the same levelof catalytic activity as microplasmin and miniplasmin, respectively.Thus, all variants of microplasmin and miniplasmin are required tocontain amino acids 603-741 of SEQ ID NO:10, which contains thecatalytic triad of the plasmin serine protease domain namely, His₆₀₃,Asp₆₄₆ and Ser₇₄₁ of human plasminogen. In one embodiment a variant ofminiplasmin includes proteins containing one or more amino aciddeletions in amino acids 444-791 of human plasminogen, wherein theresulting protein possesses serine protease catalytic activity. Inanother embodiment, a variant of microplasmin includes proteinscontaining one or more deletions in amino acids 543-791 of humanplasminogen, wherein the resulting protein possesses serine proteasecatalytic activity. In yet another embodiment, a variant of microplasminincludes proteins containing one or more deletions in amino acids562-791 of human plasminogen, wherein the resulting protein possessesserine protease catalytic activity. The deletions can be at theN-terminus, C-terminus or at an internal location of amino acids444-791, 543-791 and 562-791 of SEQ ID NO:10, respectively; however, allvariants of microplasmin and miniplasmin are required to contain aminoacids 603-741 of SEQ ID NO:10. Variants of microplasmin and miniplasminalso include, but are not limited to, amino acid insertions and/orsubstitutions in these proteins. It is envisioned that amino acidsubstitutions made in microplasmin or miniplasmin are preferablyconservative substitutions. Any variant of microplasmin and miniplasminor any other TPCD can be prepared by recombinant methods and activatedto the active plasmin form with a plasminogen activator. Alternatively,variants of microplasmin and miniplasmin or any other TPCD can beprepared by any other means well known in the art such as, but notlimited to, digestion of human plasminogen with elastase or partialreduction and alkylation of plasmin, microplasmin or miniplasmin. Thesevariants of microplasmin, miniplasmin, or for that matter any TPCD, canbe assayed for serine protease catalytic activity using the chromogenicsubstrate S2403 or any other chromogenic substrate. In addition, thevariants of microplasmin, miniplasmin or any other TPCD can be testedfor their ability to induce PVD and/or effect vitreous liquefaction byinjecting different doses of the variant in any balanced saline solutioninto porcine, feline or post-mortem human eyes. If a TPCD can induce PVDand/or effect vitreous liquefaction in any of these eyes, that TPCD isconsidered to be useful for treating eye disorders of mammals.Preferably, the TPCD does not result in toxicity to the injected eye.Non-limiting examples of variants of microplasmin are provided in Table1.

TABLE 1 Non-limiting Examples of Variants of Microplasmin The variantsof microplasmin listed below correspond to the amino acid sequence andnumbering of human plasminogen, which consists of amino acids 1-791(see, FIG. 1, SEQ ID NO: 10). 542-741, 542-742, 542-743, 542-744,542-745, 542-746, 542-747, 542-748, 542-749, 542-750, 542-751, 542-752,542-753, 542-754, 542-755, 542-756, 542-757, 542-758, 542-759, 542-760,542-761, 542-762, 542-763, 542-764, 542-765, 542-766, 542-767, 542-768,542-769, 542-770, 542-771, 542-772, 542-773, 542-774, 542-775, 542-776,542-777, 542-778, 542-779, 542-780, 542-781, 542-782, 542-783, 542-784,542-785, 542-786, 542-787, 542-788, 542-789, 542-790, 542-791; 543-741,543-742, 543-743, 543-744, 543-745, 543-746, 543-747, 543-748, 543-749,543-750, 543-751, 543-752, 543-753, 543-754, 543-755, 543-756, 543-757,543-758, 543-759, 543-760, 543-761, 543-762, 543-763, 543-764, 543-765,543-766, 543-767, 543-768, 543-769, 543-770, 543-771, 543-772, 543-773,543-774, 543-775, 543-776, 543-777, 543-778, 543-779, 543-780, 543-781,543-782, 543-783, 543-784, 543-785, 543-786, 543-787, 543-788, 543-789,543-790, 543-791; 544-741, 544-742, 544-743, 544-744, 544-745, 544-746,544-747, 544-748, 544-749, 544-750, 544-751, 544-752, 544-753, 544-754,544-755, 544-756, 544-757, 544-758, 544-759, 544-760, 544-761, 544-762,544-763, 544-764, 544-765, 544-766, 544-767, 544-768, 544-769, 544-770,544-771, 544-772, 544-773, 544-774, 544-775, 544-776, 544-777, 544-778,544-779, 544-780, 544-781, 544-782, 544-783, 544-784, 544-785, 544-786,544-787, 544-788, 544-789, 544-790, 544-791; 545-741, 545-742, 545-743,545-744, 545-745, 545-746, 545-747, 545-748, 545-749, 545-750, 545-751,545-752, 545-753, 545-754, 545-755, 545-756, 545-757, 545-758, 545-759,545-760, 545-761, 545-762, 545-763, 545-764, 545-765, 545-766, 545-767,545-768, 545-769, 545-770, 545-771, 545-772, 545-773, 545-774, 545-775,545-776, 545-777, 545-778, 545-779, 545-780, 545-781, 545-782, 545-783,545-784, 545-785, 545-786, 545-787, 545-788, 545-789, 545-790, 545-791;546-741, 546-742, 546-743, 546-744, 546-745, 546-746, 546-747, 546-748,546-749, 546-750, 546-751, 546-752, 546-753, 546-754, 546-755, 546-756,546-757, 546-758, 546-759, 546-760, 546-761, 546-762, 546-763, 546-764,546-765, 546-766, 546-767, 546-768, 546-769, 546-770, 546-771, 546-772,546-773, 546-774, 546-775, 546-776, 546-777, 546-778, 546-779, 546-780,546-781, 546-782, 546-783, 546-784, 546-785, 546-786, 546-787, 546-788,546-789, 546-790, 546-791; 547-741, 547-742, 547-743, 547-744, 547-745,547-746, 547-747, 547-748, 547-749, 547-750, 547-751, 547-752, 547-753,547-754, 547-755, 547-756, 547-757, 547-758, 547-759, 547-760, 547-761,547-762, 547-763, 547-764, 547-765, 547-766, 547-767, 547-768, 547-769,547-770, 547-771, 547-772, 547-773, 547-774, 547-775, 547-776, 547-777,547-778, 547-779, 547-780, 547-781, 547-782, 547-783, 547-784, 547-785,547-786, 547-787, 547-788, 547-789, 547-790, 547-791; 548-741, 548-742,548-743, 548-744, 548-745, 548-746, 548-747, 548-748, 548-749, 548-750,548-751, 548-752, 548-753, 548-754, 548-755, 548-756, 548-757, 548-758,548-759, 548-760, 548-761, 548-762, 548-763, 548-764, 548-765, 548-766,548-767, 548-768, 548-769, 548-770, 548-771, 548-772, 548-773, 548-774,548-775, 548-776, 548-777, 548-778, 548-779, 548-780, 548-781, 548-782,548-783, 548-784, 548-785, 548-786, 548-787, 548-788, 548-789, 548-790,548-791; 549-741, 549-742, 549-743, 549-744, 549-745, 549-746, 549-747,549-748, 549-749, 549-750, 549-751, 549-752, 549-753, 549-754, 549-755,549-756, 549-757, 549-758, 549-759, 549-760, 549-761, 549-762, 549-763,549-764, 549-765, 549-766, 549-767, 549-768, 549-769, 549-770, 549-771,549-772, 549-773, 549-774, 549-775, 549-776, 549-777, 549-778, 549-779,549-780, 549-781, 549-782, 549-783, 549-784, 549-785, 549-786, 549-787,549-788, 549-789, 549-790, 549-791; 550-741, 550-742, 550-743, 550-744,550-745, 550-746, 550-747, 550-748, 550-749, 550-750, 550-751, 550-752,550-753, 550-754, 550-755, 550-756, 550-757, 550-758, 550-759, 550-760,550-761, 550-762, 550-763, 550-764, 550-765, 550-766, 550-767, 550-768,550-769, 550-770, 550-771, 550-772, 550-773, 550-774, 550-775, 550-776,550-777, 550-778, 550-779, 550-780, 550-781, 550-782, 550-783, 550-784,550-785, 550-786, 550-787, 550-788, 550-789, 550-790, 550-791; 551-741,551-742, 551-743, 551-744, 551-745, 551-746, 551-747, 551-748, 551-749,551-750, 551-751, 551-752, 551-753, 551-754, 551-755, 551-756, 551-757,551-758, 551-759, 551-760, 551-761, 551-762, 551-763, 551-764, 551-765,551-766, 551-767, 551-768, 551-769, 551-770, 551-771, 551-772, 551-773,551-774, 551-775, 551-776, 551-777, 551-778, 551-779, 551-780, 551-781,551-782, 551-783, 551-784, 551-785, 551-786, 551-787, 551-788, 551-789,551-790, 551-791; 552-741, 552-742, 552-743, 552-744, 552-745, 552-746,552-747, 552-748, 552-749, 552-750, 552-751, 552-752, 552-753, 552-754,552-755, 552-756, 552-757, 552-758, 552-759, 552-760, 552-761, 552-762,552-763, 552-764, 552-765, 552-766, 552-767, 552-768, 552-769, 552-770,552-771, 552-772, 552-773, 552-774, 552-775, 552-776, 552-777, 552-778,552-779, 552-780, 552-781, 552-782, 552-783, 552-784, 552-785, 552-786,552-787, 552-788, 552-789, 552-790, 552-791; 553-741, 553-742, 553-743,553-744, 553-745, 553-746, 553-747, 553-748, 553-749, 553-750, 553-751,553-752, 553-753, 553-754, 553-755, 553-756, 553-757, 553-758, 553-759,553-760, 553-761, 553-762, 553-763, 553-764, 553-765, 553-766, 553-767,553-768, 553-769, 553-770, 553-771, 553-772, 553-773, 553-774, 553-775,553-776, 553-777, 553-778, 553-779, 553-780, 553-781, 553-782, 553-783,553-784, 553-785, 553-786, 553-787, 553-788, 553-789, 553-790, 553-791;554-741, 554-742, 554-743, 554-744, 554-745, 554-746, 554-747, 554-748,554-749, 554-750, 554-751, 554-752, 554-753, 554-754, 554-755, 554-756,554-757, 554-758, 554-759, 554-760, 554-761, 554-762, 554-763, 554-764,554-765, 554-766, 554-767, 554-768, 554-769, 554-770, 554-771, 554-772,554-773, 554-774, 554-775, 554-776, 554-777, 554-778, 554-779, 554-780,5544-781, 554-782, 554-783, 554-784, 554-785, 554-786, 554-787, 554-788,554-789, 554-790, 554-791; 555-741, 555-742, 555-743, 555-744, 555-745,555-746, 555-747, 555-748, 555-749, 555-750, 555-751, 555-752, 555-753,555-754, 555-755, 555-756, 555-757, 555-758, 555-759, 555-760, 555-761,555-762, 555-763, 555-764, 555-765, 555-766, 555-767, 555-768, 555-769,555-770, 555-771, 555-772, 555-773, 555-774, 555-775, 555-776, 555-777,555-778, 555-779, 555-780, 555-781, 555-782, 555-783, 555-784, 555-785,555-786, 555-787, 555-788, 555-789, 555-790, 555-791; 556-741, 556-742,556-743, 556-744, 556-745, 556-746, 556-747, 556-748, 556-749, 556-750,556-751, 556-752, 556-753, 556-754, 556-755, 556-756, 556-757, 556-758,556-759, 556-760, 556-761, 556-762, 556-763, 556-764, 556-765, 556-766,556-767, 556-768, 556-769, 556-770, 556-771, 556-772, 556-773, 556-774,556-775, 556-776, 556-777, 556-778, 556-779, 556-780, 556-781, 556-782,556-783, 556-784, 556-785, 556-786, 556-787, 556-788, 556-789, 556-790,556-791; 557-741, 557-742, 557-743, 557-744, 557-745, 557-746, 557-747,557-748, 557-749, 557-750, 557-751, 557-752, 557-753, 557-754, 557-755,557-756, 557-757, 557-758, 557-759, 557-760, 557-761, 557-762, 557-763,557-764, 557-765, 557-766, 557-767, 557-768, 557-769, 557-770, 557-771,557-772, 557-773, 557-774, 557-775, 557-776, 557-777, 557-778, 557-779,557-780, 557-781, 557-782, 557-783, 557-784, 557-785, 557-786, 557-787,557-788, 557-789, 557-790, 557-791; 558-741, 558-742, 558-743, 558-744,558-745, 558-746, 558-747, 558-748, 558-749, 558-750, 558-751, 558-752,558-753, 558-754, 558-755, 558-756, 558-757, 558-758, 558-759, 558-760,558-761, 558-762, 558-763, 558-764, 558-765, 558-766, 558-767, 558-768,558-769, 558-770, 558-771, 558-772, 558-773, 558-774, 558-775, 558-776,558-777, 558-778, 558-779, 558-780, 558-781, 558-782, 558-783, 558-784,558-785, 558-786, 558-787, 558-788, 558-789, 558-790, 558-791; 559-741,559-742, 559-743, 559-744, 559-745, 559-746, 559-747, 559-748, 559-749,559-750, 559-751, 559-752, 559-753, 559-754, 559-755, 559-756, 559-757,559-758, 559-759, 559-760, 559-761, 559-762, 559-763, 559-764, 559-765,559-766, 559-767, 559-768, 559-769, 559-770, 559-771, 559-772, 559-773,559-774, 559-775, 559-776, 559-777, 559-778, 559-779, 559-780, 559-781,559-782, 559-783, 559-784, 559-785, 559-786, 559-787, 559-788, 559-789,559-790, 559-791; 560-741, 560-742, 560-743, 560-744, 560-745, 560-746,560-747, 560-748, 560-749, 560-750, 560-751, 560-752, 560-753, 560-754,560-755, 560-756, 560-757, 560-758, 560-759, 560-760, 560-761, 560-762,560-763, 560-764, 560-765, 560-766, 560-767, 560-768, 560-769, 560-770,560-771, 560-772, 560-773, 560-774, 560-775, 560-776, 560-777, 560-778,560-779, 560-780, 560-781, 560-782, 560-783, 560-784, 560-785, 560-786,560-787, 560-788, 560-789, 560-790, 560-791; 561-741, 561-742, 561-743,561-744, 561-745, 561-746, 561-747, 561-748, 561-749, 561-750, 561-751,561-752, 561-753, 561-754, 561-755, 561-756, 561-757, 561-758, 561-759,561-760, 561-761, 561-762, 561-763, 561-764, 561-765, 561-766, 561-767,561-768, 561-769, 561-770, 561-771, 561-772, 561-773, 561-774, 561-775,561-776, 561-777, 561-778, 561-779, 561-780, 561-781, 561-782, 561-783,561-784, 561-785, 561-786, 561-787, 561-788, 561-789, 561-790, 561-791;562-741, 562-742, 562-743, 562-744, 562-745, 562-746, 562-747, 562-748,562-749, 562-750, 562-751, 562-752, 562-753, 562-754, 562-755, 562-756,562-757, 562-758, 562-759, 562-760, 562-761, 562-762, 562-763, 562-764,562-765, 562-766, 562-767, 562-768, 562-769, 562-770, 562-771, 562-772,562-773, 562-774, 562-775, 562-776, 562-777, 562-778, 562-779, 562-780,562-781, 562-782, 562-783, 562-784, 562-785, 562-786, 562-787, 562-788,562-789, 562-790, 562-791; 563-741, 563-742, 563-743, 563-744, 563-745,563-746, 563-747, 563-748, 563-749, 563-750, 563-751, 563-752, 563-753,563-754, 563-755, 563-756, 563-757, 563-758, 563-759, 563-760, 563-761,563-762, 563-763, 563-764, 563-765, 563-766, 563-767, 563-768, 563-769,563-770, 563-771, 563-772, 563-773, 563-774, 563-775, 563-776, 563-777,563-778, 563-779, 563-780, 563-781, 563-782, 563-783, 563-784, 563-785,563-786, 563-787, 563-788, 563-789, 563-790, 563-791; 564-741, 564-742,564-743, 564-744, 564-745, 564-746, 564-747, 564-748, 564-749, 564-750,564-751, 564-752, 564-753, 564-754, 564-755, 564-756, 564-757, 564-758,564-759, 564-760, 564-761, 564-762, 564-763, 564-764, 564-765, 564-766,564-767, 564-768, 564-769, 564-770, 564-771, 564-772, 564-773, 564-774,564-775, 564-776, 564-777, 564-778, 564-779, 564-780, 564-781, 564-782,564-783, 564-784, 564-785, 564-786, 564-787, 564-788, 564-789, 564-790,564-791; 565-741, 565-742, 565-743, 565-744, 565-745, 565-746, 565-747,565-748, 565-749, 565-750, 565-751, 565-752, 565-753, 565-754, 565-755,565-756, 565-757, 565-758, 565-759, 565-760, 565-761, 565-762, 565-763,565-764, 565-765, 565-766, 565-767, 565-768, 565-769, 565-770, 565-771,565-772, 565-773, 565-774, 565-775, 565-776, 565-777, 565-778, 565-779,565-780, 565-781, 565-782, 565-783, 565-784, 565-785, 565-786, 565-787,565-788, 565-789, 565-790, 565-791; 566-741, 566-742, 566-743, 566-744,566-745, 566-746, 566-747, 566-748, 566-749, 566-750, 566-751, 566-752,566-753, 566-754, 566-755, 566-756, 566-757, 566-758, 566-759, 566-760,566-761, 566-762, 566-763, 566-764, 566-765, 566-766, 566-767, 566-768,566-769, 566-770, 566-771, 566-772, 566-773, 566-774, 566-775, 566-776,566-777, 566-778, 566-779, 566-780, 566-781, 566-782, 566-783, 566-784,566-785, 566-786, 566-787, 566-788, 566-789, 566-790, 566-791; 567-741,567-742, 567-743, 567-744, 567-745, 567-746, 567-747, 567-748, 567-749,567-750, 567-751, 567-752, 567-753, 567-754, 567-755, 567-756, 567-757,567-758, 567-759, 567-760, 567-761, 567-762, 567-763, 567-764, 567-765,567-766, 567-767, 567-768, 567-769, 567-770, 567-771, 567-772, 567-773,567-774, 567-775, 567-776, 567-777, 567-778, 567-779, 567-780, 567-781,567-782, 567-783, 567-784, 567-785, 567-786, 567-787, 567-788, 567-789,567-790, 567-791; 568-741, 568-742, 568-743, 568-744, 568-745, 568-746,568-747, 568-748, 568-749, 568-750, 568-751, 568-752, 568-753, 568-754,568-755, 568-756, 568-757, 568-758, 568-759, 568-760, 568-761, 568-762,568-763, 568-764, 568-765, 568-766, 568-767, 568-768, 568-769, 568-770,568-771, 568-772, 568-773, 568-774, 568-775, 568-776, 568-777, 568-778,568-779, 568-780, 568-781, 568-782, 568-783, 568-784, 568-785, 568-786,568-787, 568-788, 568-789, 568-790, 568-791; 569-741, 569-742, 569-743,569-744, 569-745, 569-746, 569-747, 569-748, 569-749, 569-750, 569-751,569-752, 569-753, 569-754, 569-755, 569-756, 569-757, 569-758, 569-759,569-760, 569-761, 569-762, 569-763, 569-764, 569-765, 569-766, 569-767,569-768, 569-769, 569-770, 569-771, 569-772, 569-773, 569-774, 569-775,569-776, 569-777, 569-778, 569-779, 569-780, 569-781, 569-782, 569-783,569-784, 569-785, 569-786, 569-787, 569-788, 569-789, 569-790, 569-791;570-741, 570-742, 570-743, 570-744, 570-745, 570-746, 570-747, 570-748,570-749, 570-750, 570-751, 570-752, 570-753, 570-754, 570-755, 570-756,570-757, 570-758, 570-759, 570-760, 570-761, 570-762, 570-763, 570-764,570-765, 570-766, 570-767, 570-768, 570-769, 570-770, 570-771, 570-772,570-773, 570-774, 570-775, 570-776, 570-777, 570-778, 570-779, 570-780,570-781, 570-782, 570-783, 570-784, 570-785, 570-786, 570-787, 570-788,570-789, 570-790, 570-791; 571-741, 571-742, 571-743, 571-744, 571-745,571-746, 571-747, 571-748, 571-749, 571-750, 571-751, 571-752, 571-753,571-754, 571-755, 571-756, 571-757, 571-758, 571-759, 571-760, 571-761,571-762, 571-763, 571-764, 571-765, 571-766, 571-767, 571-768, 571-769,571-770, 571-771, 571-772, 571-773, 571-774, 571-775, 571-776, 571-777,571-778, 571-779, 571-780, 571-781, 571-782, 571-783, 571-784, 571-785,571-786, 571-787, 571-788, 571-789, 571-790, 571-791; 572-741, 572-742,572-743, 572-744, 572-745, 572-746, 572-747, 572-748, 572-749, 572-750,572-751, 572-752, 572-753, 572-754, 572-755, 572-756, 572-757, 572-758,572-759, 572-760, 572-761, 572-762, 572-763, 572-764, 572-765, 572-766,572-767, 572-768, 572-769, 572-770, 572-771, 572-772, 572-773, 572-774,572-775, 572-776, 572-777, 572-778, 572-779, 572-780, 572-781, 572-782,572-783, 572-784, 572-785, 572-786, 572-787, 572-788, 572-789, 572-790,572-791; 573-741, 573-742, 573-743, 573-744, 573-745, 573-746, 573-747,573-748, 573-749, 573-750, 573-751, 573-752, 573-753, 573-754, 573-755,573-756, 573-757, 573-758, 573-759, 573-760, 573-761, 573-762, 573-763,573-764, 573-765, 573-766, 573-767, 573-768, 573-769, 573-770, 573-771,573-772, 573-773, 573-774, 573-775, 573-776, 573-777, 573-778, 573-779,573-780, 573-781, 573-782, 573-783, 573-784, 573-785, 573-786, 573-787,573-788, 573-789, 573-790, 573-791; 574-741, 574-742, 574-743, 574-744,574-745, 574-746, 574-747, 574-748, 574-749, 574-750, 574-751, 574-752,574-753, 574-754, 574-755, 574-756, 574-757, 574-758, 574-759, 574-760,574-761, 574-762, 574-763, 574-764, 574-765, 574-766, 574-767, 574-768,574-769, 574-770, 574-771, 574-772, 574-773, 574-774, 574-775, 574-776,574-777, 574-778, 574-779, 574-780, 574-781, 574-782, 574-783, 574-784,574-785, 574-786, 574-787, 574-788, 574-789, 574-790, 574-791; 575-741,575-742, 575-743, 575-744, 575-745, 575-746, 575-747, 575-748, 575-749,575-750, 575-751, 575-752, 575-753, 575-754, 575-755, 575-756, 575-757,575-758, 575-759, 575-760, 575-761, 575-762, 575-763, 575-764, 575-765,575-766, 575-767, 575-768, 575-769, 575-770, 575-771, 575-772, 575-773,575-774, 575-775, 575-776, 575-777, 575-778, 575-779, 575-780, 575-781,575-782, 575-783, 575-784, 575-785, 575-786, 575-787, 575-788, 575-789,575-790, 575-791; 576-741, 576-742, 576-743, 576-744, 576-745, 576-746,576-747, 576-748, 576-749, 576-750, 576-751, 576-752, 576-753, 576-754,576-755, 576-756, 576-757, 576-758, 576-759, 576-760, 576-761, 576-762,576-763, 576-764, 576-765, 576-766, 576-767, 576-768, 576-769, 576-770,576-771, 576-772, 576-773, 576-774, 576-775, 576-776, 576-777, 576-778,576-779, 576-780, 576-781, 576-782, 576-783, 576-784, 576-785, 576-786,576-787, 576-788, 576-789, 576-790, 576-791; 577-741, 577-742, 577-743,577-744, 577-745, 577-746, 577-747, 577-748, 577-749, 577-750, 577-751,577-752, 577-753, 577-754, 577-755, 577-756, 577-757, 577-758, 577-759,577-760, 577-761, 577-762, 577-763, 577-764, 577-765, 577-766, 577-767,577-768, 577-769, 577-770, 577-771, 577-772, 577-773, 577-774, 577-775,577-776, 577-777, 577-778, 577-779, 577-780, 577-781, 577-782, 577-783,577-784, 577-785, 577-786, 577-787, 577-788, 577-789, 577-790, 577-791;578-741, 578-742, 578-743, 578-744, 578-745, 578-746, 578-747, 578-748,578-749, 578-750, 578-751, 578-752, 578-753, 578-754, 578-755, 578-756,578-757, 578-758, 578-759, 578-760, 578-761, 578-762, 578-763, 578-764,578-765, 578-766, 578-767, 578-768, 578-769, 578-770, 578-771, 578-772,578-773, 578-774, 578-775, 578-776, 578-777, 578-778, 578-779, 578-780,578-781, 578-782, 578-783, 578-784, 578-785, 578-786, 578-787, 578-788,578-789, 578-790, 578-791; 579-741, 579-742, 579-743, 579-744, 579-745,579-746, 579-747, 579-748, 579-749, 579-750, 579-751, 579-752, 579-753,579-754, 579-755, 579-756, 579-757, 579-758, 579-759, 579-760, 579-761,579-762, 579-763, 579-764, 579-765, 579-766, 579-767, 579-768, 579-769,579-770, 579-771, 579-772, 579-773, 579-774, 579-775, 579-776, 579-777,579-778, 579-779, 579-780, 579-781, 579-782, 579-783, 579-784, 579-785,579-786, 579-787, 579-788, 579-789, 579-790, 579-791; 580-741, 580-742,580-743, 580-744, 580-745, 580-746, 580-747, 580-748, 580-749, 580-750,580-751, 580-752, 580-753, 580-754, 580-755, 580-756, 580-757, 580-758,580-759, 580-760, 580-761, 580-762, 580-763, 580-764, 580-765, 580-766,580-767, 580-768, 580-769, 580-770, 580-771, 580-772, 580-773, 580-774,580-775, 580-776, 580-777, 580-778, 580-779, 580-780, 580-781, 580-782,580-783, 580-784, 580-785, 580-786, 580-787, 580-788, 580-789, 580-790,580-791; 581-741, 581-742, 581-743, 581-744, 581-745, 581-746, 581-747,581-748, 581-749, 581-750, 581-751, 581-752, 581-753, 581-754, 581-755,581-756, 581-757, 581-758, 581-759, 581-760, 581-761, 581-762, 581-763,581-764, 581-765, 581-766, 581-767, 581-768, 581-769, 581-770, 581-771,581-772, 581-773, 581-774, 581-775, 581-776, 581-777, 581-778, 581-779,581-780, 581-781, 581-782, 581-783, 581-784, 581-785, 581-786, 581-787,581-788, 581-789, 581-790, 581-791; 582-741, 582-742, 582-743, 582-744,582-745, 582-746, 582-747, 582-748, 582-749, 582-750, 582-751, 582-752,582-753, 582-754, 582-755, 582-756, 582-757, 582-758, 582-759, 582-760,582-761, 582-762, 582-763, 582-764, 582-765, 582-766, 582-767, 582-768,582-769, 582-770, 582-771, 582-772, 582-773, 582-774, 582-775, 582-776,582-777, 582-778, 582-779, 582-780, 582-781, 582-782, 582-783, 582-784,582-785, 582-786, 582-787, 582-788, 582-789, 582-790, 582-791; 583-741,583-742, 583-743, 583-744, 583-745, 583-746, 583-747, 583-748, 583-749,583-750, 583-751, 583-752, 583-753, 583-754, 583-755, 583-756, 583-757,583-758, 583-759, 583-760, 583-761, 583-762, 583-763, 583-764, 583-765,583-766, 583-767, 583-768, 583-769, 583-770, 583-771, 583-772, 583-773,583-774, 583-775, 583-776, 583-777, 583-778, 583-779, 583-780, 583-781,583-782, 583-783, 583-784, 583-785, 583-786, 583-787, 583-788, 583-789,583-790, 583-791; 584-741, 584-742, 584-743, 584-744, 584-745, 584-746,584-747, 584-748, 584-749, 584-750, 584-751, 584-752, 584-753, 584-754,584-755, 584-756, 584-757, 584-758, 584-759, 584-760, 584-761, 584-762,584-763, 584-764, 584-765, 584-766, 584-767, 584-768, 584-769, 584-770,584-771, 584-772, 584-773, 584-774, 584-775, 584-776, 584-777, 584-778,584-779, 584-780, 584-781, 584-782, 584-783, 584-784, 584-785, 584-786,584-787, 584-788, 584-789, 584-790, 584-791; 585-741, 585-742, 585-743,585-744, 585-745, 585-746, 585-747, 585-748, 585-749, 585-750, 585-751,585-752, 585-753, 585-754, 585-755, 585-756, 585-757, 585-758, 585-759,585-760, 585-761, 585-762, 585-763, 585-764, 585-765, 585-766, 585-767,585-768, 585-769, 585-770, 585-771, 585-772, 585-773, 585-774, 585-775,585-776, 585-777, 585-778, 585-779, 585-780, 585-781, 585-782, 585-783,585-784, 585-785, 585-786, 585-787, 585-788, 585-789, 585-790, 585-791;586-741, 586-742, 586-743, 586-744, 586-745, 586-746, 586-747, 586-748,586-749, 586-750, 586-751, 586-752, 586-753, 586-754, 586-755, 586-756,586-757, 586-758, 586-759, 586-760, 586-761, 586-762, 586-763, 586-764,586-765, 586-766, 586-767, 586-768, 586-769, 586-770, 586-771, 586-772,586-773, 586-774, 586-775, 586-776, 586-777, 586-778, 586-779, 586-780,586-781, 586-782, 586-783, 586-784, 586-785, 586-786, 586-787, 586-788,586-789, 586-790, 586-791; 587-741, 587-742, 587-743, 587-744, 587-745,587-746, 587-747, 587-748, 587-749, 587-750, 587-751, 587-752, 587-753,587-754, 587-755, 587-756, 587-757, 587-758, 587-759, 587-760, 587-761,587-762, 587-763, 587-764, 587-765, 587-766, 587-767, 587-768, 587-769,587-770, 587-771, 587-772, 587-773, 587-774, 587-775, 587-776, 587-777,587-778, 587-779, 587-780, 587-781, 587-782, 587-783, 587-784, 587-785,587-786, 587-787, 587-788, 587-789, 587-790, 587-791; 588-741, 588-742,588-743, 588-744, 588-745, 588-746, 588-747, 588-748, 588-749, 588-750,588-751, 588-752, 588-753, 588-754, 588-755, 588-756, 588-757, 588-758,588-759, 588-760, 588-761, 588-762, 588-763, 588-764, 588-765, 588-766,588-767, 588-768, 588-769, 588-770, 588-771, 588-772, 588-773, 588-774,588-775, 588-776, 588-777, 588-778, 588-779, 588-780, 588-781, 588-782,588-783, 588-784, 588-785, 588-786, 588-787, 588-788, 588-789, 588-790,588-791; 589-741, 589-742, 589-743, 589-744, 589-745, 589-746, 589-747,589-748, 589-749, 589-750, 589-751, 589-752, 589-753, 589-754, 589-755,589-756, 589-757, 589-758, 589-759, 589-760, 589-761, 589-762, 589-763,589-764, 589-765, 589-766, 589-767, 589-768, 589-769, 589-770, 589-771,589-772, 589-773, 589-774, 589-775, 589-776, 589-777, 589-778, 589-779,589-780, 589-781, 589-782, 589-783, 589-784, 589-785, 589-786, 589-787,589-788, 589-789, 589-790, 589-791; 590-741, 590-742, 590-743, 590-744,590-745, 590-746, 590-747, 590-748, 590-749, 590-750, 590-751, 590-752,590-753, 590-754, 590-755, 590-756, 590-757, 590-758, 590-759, 590-760,590-761, 590-762, 590-763, 590-764, 590-765, 590-766, 590-767, 590-768,590-769, 590-770, 590-771, 590-772, 590-773, 590-774, 590-775, 590-776,590-777, 590-778, 590-779, 590-780, 590-781, 590-782, 590-783, 590-784,590-785, 590-786, 590-787, 590-788, 590-789, 590-790, 590-791; 591-741,591-742, 591-743, 591-744, 591-745, 591-746, 591-747, 591-748, 591-749,591-750, 591-751, 591-752, 591-753, 591-754, 591-755, 591-756, 591-757,591-758, 591-759, 591-760, 591-761, 591-762, 591-763, 591-764, 591-765,591-766, 591-767, 591-768, 591-769, 591-770, 591-771, 591-772, 591-773,591-774, 591-775, 591-776, 591-777, 591-778, 591-779, 591-780, 591-781,591-782, 591-783, 591-784, 591-785, 591-786, 591-787, 591-788, 591-789,591-790, 591-791; 592-741, 592-742, 592-743, 592-744, 592-745, 592-746,592-747, 592-748, 592-749, 592-750, 592-751, 592-752, 592-753, 592-754,592-755, 592-756, 592-757, 592-758, 592-759, 592-760, 592-761, 592-762,592-763, 592-764, 592-765, 592-766, 592-767, 592-768, 592-769, 592-770,592-771, 592-772, 592-773, 592-774, 592-775, 592-776, 592-777, 592-778,592-779, 592-780, 592-781, 592-782, 592-783, 592-784, 592-785, 592-786,592-787, 592-788, 592-789, 592-790, 592-791; 593-741, 593-742, 593-743,593-744, 593-745, 593-746, 593-747, 593-748, 593-749, 593-750, 593-751,593-752, 593-753, 593-754, 593-755, 593-756, 593-757, 593-758, 593-759,593-760, 593-761, 593-762, 593-763, 593-764, 593-765, 593-766, 593-767,593-768, 593-769, 593-770, 593-771, 593-772, 593-773, 593-774, 593-775,593-776, 593-777, 593-778, 593-779, 593-780, 593-781, 593-782, 593-783,593-784, 593-785, 593-786, 593-787, 593-788, 593-789, 593-790, 593-791;594-741, 594-742, 594-743, 594-744, 594-745, 594-746, 594-747, 594-748,594-749, 594-750, 594-751, 594-752, 594-753, 594-754, 594-755, 594-756,594-757, 594-758, 594-759, 594-760, 594-761, 594-762, 594-763, 594-764,594-765, 594-766, 594-767, 594-768, 594-769, 594-770, 594-771, 594-772,594-773, 594-774, 594-775, 594-776, 594-777, 594-778, 594-779, 594-780,594-781, 594-782, 594-783, 594-784, 594-785, 594-786, 594-787, 594-788,594-789, 594-790, 594-791; 595-741, 595-742, 595-743, 595-744, 595-745,595-746, 595-747, 595-748, 595-749, 595-750, 595-751, 595-752, 595-753,595-754, 595-755, 595-756, 595-757, 595-758, 595-759, 595-760, 595-761,595-762, 595-763, 595-764, 595-765, 595-766, 595-767, 595-768, 595-769,595-770, 595-771, 595-772, 595-773, 595-774, 595-775, 595-776, 595-777,595-778, 595-779, 595-780, 595-781, 595-782, 595-783, 595-784, 595-785,595-786, 595-787, 595-788, 595-789, 595-790, 595-791; 596-741, 596-742,596-743, 596-744, 596-745, 596-746, 596-747, 596-748, 596-749, 596-750,596-751, 596-752, 596-753, 596-754, 596-755, 596-756, 596-757, 596-758,596-759, 596-760, 596-761, 596-762, 596-763, 596-764, 596-765, 596-766,596-767, 596-768, 596-769, 596-770, 596-771, 596-772, 596-773, 596-774,596-775, 596-776, 596-777, 596-778, 596-779, 596-780, 596-781, 596-782,596-783, 596-784, 596-785, 596-786, 596-787, 596-788, 596-789, 596-790,596-791; 597-741, 597-742, 597-743, 597-744, 597-745, 597-746, 597-747,597-748, 597-749, 597-750, 597-751, 597-752, 597-753, 597-754, 597-755,597-756, 597-757, 597-758, 597-759, 597-760, 597-761, 597-762, 597-763,597-764, 597-765, 597-766, 597-767, 597-768, 597-769, 597-770, 597-771,597-772, 597-773, 597-774, 597-775, 597-776, 597-777, 597-778, 597-779,597-780, 597-781, 597-782, 597-783, 597-784, 597-785, 597-786, 597-787,597-788, 597-789, 597-790, 597-791; 598-741, 598-742, 598-743, 598-744,598-745, 598-746, 598-747, 598-748, 598-749, 598-750, 598-751, 598-752,598-753, 598-754, 598-755, 598-756, 598-757, 598-758, 598-759, 598-760,598-761, 598-762, 598-763, 598-764, 598-765, 598-766, 598-767, 598-768,598-769, 598-770, 598-771, 598-772, 598-773, 598-774, 598-775, 598-776,598-777, 598-778, 598-779, 598-780, 598-781, 598-782, 598-783, 598-784,598-785, 598-786, 598-787, 598-788, 598-789, 598-790, 598-791; 599-741,599-742, 599-743, 599-744, 599-745, 599-746, 599-747, 599-748, 599-749,599-750, 599-751, 599-752, 599-753, 599-754, 599-755, 599-756, 599-757,599-758, 599-759, 599-760, 599-761, 599-762, 599-763, 599-764, 599-765,599-766, 599-767, 599-768, 599-769, 599-770, 599-771, 599-772, 599-773,599-774, 599-775, 599-776, 599-777, 599-778, 599-779, 599-780, 599-781,599-782, 599-783, 599-784, 599-785, 599-786, 599-787, 599-788, 599-789,599-790, 599-791; 600-741, 600-742, 600-743, 600-744, 600-745, 600-746,600-747, 600-748, 600-749, 600-750, 600-751, 600-752, 600-753, 600-754,600-755, 600-756, 600-757, 600-758, 600-759, 600-760, 600-761, 600-762,600-763, 600-764, 600-765, 600-766, 600-767, 600-768, 600-769, 600-770,600-771, 600-772, 600-773, 600-774, 600-775, 600-776, 600-777, 600-778,600-779, 600-780, 600-781, 600-782, 600-783, 600-784, 600-785, 600-786,600-787, 600-788, 600-789, 600-790, 600-791; 601-741, 601-742, 601-743,601-744, 601-745, 601-746, 601-747, 601-748, 601-749, 601-750, 601-751,601-752, 601-753, 601-754, 601-755, 601-756, 601-757, 601-758, 601-759,601-760, 601-761, 601-762, 601-763, 601-764, 601-765, 601-766, 601-767,601-768, 601-769, 601-770, 601-771, 601-772, 601-773, 601-774, 601-775,601-776, 601-777, 601-778, 601-779, 601-780, 601-781, 601-782, 601-783,601-784, 601-785, 601-786, 601-787, 601-788, 601-789, 601-790, 601-791;602-741, 602-742, 602-743, 602-744, 602-745, 602-746, 602-747, 602-748,602-749, 602-750, 602-751, 602-752, 602-753, 602-754, 602-755, 602-756,602-757, 602-758, 602-759, 602-760, 602-761, 602-762, 602-763, 602-764,602-765, 602-766, 602-767, 602-768, 602-769, 602-770, 602-771, 602-772,602-773, 602-774, 602-775, 602-776, 602-777, 602-778, 602-779, 602-780,602-781, 602-782, 602-783, 602-784, 602-785, 602-786, 602-787, 602-788,602-789, 602-790, 602-791

Miniplasmin and microplasmin are produced upon the activation ofminiplasminogen and microplasminogen by plasminogen activators such as,but not limited to, streptokinase, staphylokinase, tissue-typeplasminogen activator or urokinase. Miniplasminogen and microplasminogenare derived from plasminogen, which is a single chain glycoprotein thatis an important component of mammalian blood. Human plasminogen is amulti-domain protein of 791 residues (SEQ ID NO:10), composed of anN-terminal pre-activation domain, five homologous kringle domains eachof about 80 amino acids, a serine protease catalytic domain andinter-domain connecting sequences. Plasmin or plasminogen activatorscleave the peptide bonds between Arg₆₈-Met₆₉, or Lys₇₇-Lys₇₈ orLys₇₈-Val₇₉ at the N-terminal of human plasminogen, resulting in shorterproenzymes called Lys-plasminogens (for example, proteins consisting ofamino acids 69-791 or 78-791 or 79-791). Additional cleavage by theenzyme elastase removes the first four kringle domains producing theproenzyme, miniplasminogen (typically amino acids 442-791). Furthercleavage of the fifth kringle yields the proenzyme, microplasminogen(typically amino acids 543-791). The kringles of plasminogen containlysine-binding sites that mediate specific binding of plasminogen tosubstrates such as fibrin. The proenzyme forms of plasminogen areactivated to their enzymatically active form by the cleavage of thepeptide bond between Arg₅₆₁ and Val₅₆₂ to yield a disulfide bondeddouble chain form of the corresponding protein. The product ofactivation of a plasminogen protein is called a plasmin. Thus, theproduct of Lys-plasminogen activation is called Lys-plasmin, while theproducts of activation of miniplasminogen and microplasminogen, arereferred to as miniplasmin and microplasmin, respectively. Lys-plasminhas a molecular weight of about 65,000 in its unglycosylated form and amolecular weight of about 83,000 daltons in its fully glycosylated form,while miniplasmin has a molecular weight of about 38,000 daltons, andmicroplasmin has a molecular weight of about 26,500 daltons in thereduced form and about 29,000 daltons in the non-reduced form. Likeplasmin, miniplasmin and microplasmin possess catalytic activity. Anadvantage of miniplasmin and microplasmin over plasmin is their smallersize compared to plasmin. Thus, both microplasmin and miniplasmin areexpected to have faster diffusion rates in the vitreous than plasmin(Xu, J. et al., Pharmaceutical Research 17: 664-669, 2000).

In one embodiment, a TPCD has a molecular weight of less than about40,000 daltons. In another embodiment, a TPCD has a molecular weight ofbetween about 20,000 and about 30,000 daltons. In yet anotherembodiment, a TPCD has a molecular weight of about 26,500 daltons inreduced form and about 29,000 daltons in non-reduced form. In a furtherembodiment a TPCD has a molecular weight less than about 20,000 daltons.

Microplasmin can be prepared by the autolytic reaction of plasmin andplasminogen in high alkaline solution having a pH ranging from about 9.5to 11.5, as described in U.S. Pat. No. 4,774,087. Alternatively,microplasmin and miniplasmin can be prepared by recombinant methods asdescribed in PCT application WO 02/50290. Briefly, DNA encodingminiplasminogen and microplasminogen are independently cloned into ayeast expression vector (for e.g., pPICZα A secretion vector fromInvitrogen Corporation) that can be used to express these proteins inmethylotropic yeasts (e.g., Hansenula, Pichia, Candida, and Torulopsis).Yeast clones that produce proteins with the highest miniplasmin andmicroplasmin activity are selected for large-scale production. Theseclones can be grown at any scale, but typically at about a 20 liter toabout a 500 liter scale. The secreted miniplasminogen ormicroplasminogen are purified in a three-step process comprising cationexchange expanded bed chromatography, hydrophobic chromatography, andaffinity chromatography. The purified microplasminogen andminiplasminogen obtained by this process are activated to their activeforms using a molar ratio of a plasminogen activator (e.g., urokinase,streptokinase, staphylokinase, the SY162 staphylokinase variant, etc.).It should be noted that the recombinant process for producingminiplasmin and microplasmin can be extended to produce any TPCD. Anadvantage of using a recombinant TPCD compared to autologous plasminenzyme is that the recombinant proteins can be prepared from largeproduction batches resulting in enzymes of uniform activity. Becausethese proteins are of uniform activity, standardized protocols can beimplemented. A further advantage is that these proteins could be readilyavailable without the delay and other attendant problems associated withthe isolation and purification of plasmin from each patient.

The TPCD obtained by the processes described above can be concentrated,stabilized and/or lyophilized. Methods of concentrating proteins arewell known to those of ordinary skill in the art (see for example,Protein Purification Methods: A Practical Approach, Harris, E. L. V andAngal, S. (eds.), IRL Press, 1989; A Guide to Protein Isolation (SecondEdition), Clive Dennison, Kluwer Academic Publications, 2003; andProtein Methods (Second Edition), Daniel M. Bollag, Michael D. Rozycki,Stuart J. Edelstein (eds.), Wiley, 1996).

Stabilization is a method of protecting a protein from degradationand/or inactivation through the use of one or more stabilizing agents(for e.g., by contacting a TPCD with a stabilizing agent, or purifying aTPCD in the presence of a stabilizing agent). Stabilizing agents includewithout limitation, tranexamic acid, hexanoic acid, lysine, serine,threonine, methionine, glutamine, alanine, glycine, isoleucine, valine,alanine aspartic acid, polyhydric alcohol, pharmaceutically acceptablecarbohydrates, glucosamine, thiamine, niacinamide, any acidic buffercomprising citric acid, acetic acid, hydrochloric acid, carboxylic acid,lactic acid, malic acid, tartaric acid, or benzoic acid, and salts suchas sodium chloride, potassium chloride, magnesium chloride, calciumchloride, and any derivatives or combinations thereof. One advantage ofusing stabilized, recombinantly produced TPCD compared to autologousplasmin enzyme is that these proteins are more stable than autologousplasmin enzyme, which is obtained by collecting blood and, purifying,preparing and storing plasmin enzyme on a patient-by-patient basis.Unlike autologous plasmin enzyme, which needs to be used very soon afterits preparation, stabilized, recombinant TPCD can be used even after asignificant period of time from the time of purification.

Lyophilization of a TPCD of the invention can be performed immediatelyafter the concentration of the purified proteins or after stabilization.Methods of lyophilizing proteins are well known to those of ordinaryskill in the art. The lyophilized TPCD can be stored in vials (e.g.,glass) in any amount, but preferably, in amounts that can be readilyreconstituted for use.

Lyophilized microplasmin, miniplasmin, or any other TPCD, can bereconstituted in an ophthalmologically acceptable carrier prior to beingused for contacting the vitreous and/or aqueous humor. In one embodimentan ophthalmologically acceptable carrier is a sterile solvent having apH and osmolarity that is compatible with the vitreous of the subject.Nonlimiting examples of ophthalmologically acceptable carriers areisotonic saline solution, balanced salt solution (BSS) and BSS PLUS®. Abalanced salt solution typically contains: 0.64% sodium chloride, 0.075%potassium chloride, 0.048% calcium chloride dehydrate, 0.03% magnesiumchloride hexahydrate, 0.39% sodium acetate trihydrate, 0.17% sodiumcitrate dihydrate, sodium hydride/hydrochloric acid to adjust the pH,and water.

The method of contacting the vitreous and/or aqueous humor usingcompositions comprising a TPCD will depend upon the particular subject,the severity of the condition being treated and the dosage required fortherapeutic efficacy, and can be determined by a physician on apatient-by-patient basis. Any method of contacting the vitreous and/oraqueous humor that provides an effective amount of a TPCD to thevitreous and/or aqueous humor can be utilized. It should be understoodthat such contact with the vitreous and/or aqueous humor does not haveto take place simultaneously with the administration of a compositioncomprising a TPCD. The contact may be delayed or occur over an extendedperiod of time from the time of administration. One method of contactingthe vitreous and/or aqueous humor is by one or more intraocularinjections directly into the vitreous and/or aqueous humor respectively.The vitreous and/or aqueous humor can also be contacted bysub-conjunctival, intramuscular or intravenous injections. Any of theseinjections can be provided using a liquid solution comprising a TPCDaccording to procedures well known in the art. Alternatively, however,the vitreous and/or aqueous humor can be contacted with a TPCD by anyother suitable method, which results in sufficient distribution of theTPCD to the vitreous and/or aqueous humor to treat or prevent thedisorder, or a complication of a disorder, of the eye of a subject. Acomposition comprising a TPCD can also be administered by placing anintra-vitreal implantable devices including, but not limited to,OCUSERT® (Alza Corp., Palo Alto, Calif.) and VITRASERT® (Bausch andLomb, Inc., Rochester, N.Y.). The present invention also envisions thatthe vitreous and/or aqueous humor can be contacted with a TPCD using adepot, sustained release formulation, or any implantable device so thata TPCD is supplied continuously.

Dosing regimens for TPCD can be readily determined by one of ordinaryskill in the art and will vary depending on the patient and the effectsought. TPCD can be used at any dose, which brings about desirabletherapeutic effects, including but not limited to vitreous liquefaction,posterior vitreous detachment, and/or clearing of blood, toxic materialsor foreign substances from the vitreous cavity, without causingsignificant toxicity to the eye (especially the retina) or associatedanatomical structures. Additionally, a TPCD may be administered as asingle dose or in multiple doses. A typical TPCD dosage is in the rangeof about 0.005 mg to about 0.2 mg per eye. If injected, TPCD can beprovided in a delivery volume of about 0.05 ml to about 0.3 ml of asterile solvent (e.g. sterile BSS or BSS PLUS®) per eye. In thoseinstances where a vitrectomy is to be performed, the TPCD is left in thevitreous and/or aqueous humor for between about 15 and 120 minutesbefore removal of the vitreous. In one embodiment of the invention, adose of 0.125 mg of TPCD is delivered in 0.1 ml of sterile BSS or BSSPLUS® per eye. In another embodiment, a dose of 0.125 mg of TPCD isdelivered in 0.1 ml of sterile BSS or BSS PLUS® per eye for about 15-120minutes prior to vitrectomy.

The present invention also contemplates the use of compositionscomprising more than one TPCD. Accordingly, in one aspect of theinvention, the vitreous and/or aqueous humor is contacted with acomposition comprising a first TPCD and a second TPCD. In one particularembodiment of this aspect of the invention, the first and second TPCDare selected from the group consisting of miniplasmin, recombinantminiplasmin, stabilized miniplasmin, stabilized, recombinantminiplasmin, variants of miniplasmin, microplasmin, recombinantmicroplasmin, stabilized microplasmin, stabilized, recombinantmicroplasmin, variants of microplasmin, and any combinations thereof. Inanother aspect of the invention, the vitreous and/or aqueous humor iscontacted with a first composition comprising at least one TPCD and witha second composition comprising at least one TPCD. The TPCD can be thesame or different proteins and can be administered at substantially thesame time or at different times. Additionally, a TPCD can also beadministered as a composition further comprising at least one secondagent. Furthermore, the vitreous and/or aqueous humor may be contactedwith a composition comprising at least one TPCD followed by acomposition comprising at least one second agent or vice versa. This maybe necessary where the time required for each of these compositions isdifferent, i.e., where one compositions needs more time to act comparedto the other. A second agent is any protein (but not a TPCD), chemicalor other substance that is useful in treating or preventing eyedisorders, or complications of an eye disorder. Such second agents aredescribed in U.S. Pat. Nos. 4,820,516; 5,292,509; 5,866,120; 6,051,698;6,462,071; 6,596,725; and 6,610,292. Non-limiting examples of secondagents usable with the present invention include glycosaminoglycanaseenzymes such as hyaluronidases, chondroitinase ABC, chondroitinase AC,chondroitinase B, chondroitin 4-sulfatase, chondroitin 6-sulfatase andB-glucuronidase; collagenase enzymes; dispase; RGD containing peptidessuch as RGD, GRGDS, GRGDTP, Echistatin and Falvoridin; anti-integrinantibody; P2Y receptor antagonists; urea, hydroxyurea, thiourea andanti-angiogenic agents such as, but not limited to, vascular endothelialgrowth factor (VEGF) inhibitors (e.g., anti-VEGF antibodies, VEGFaptamers, soluble VEGF receptors, etc.) and placental growth factor(PlGF) inhibitors (e.g., anti-PlGF antibodies, PlGF aptamers, solubleVEGF receptors, etc.). Most of these second agents are themselvescapable of promoting vitreous liquefaction and/or inducing posteriorvitreous detachment. Anti-angiogenic second agents could be useful inpreventing neo-vascularization in the eye. Expression of VEGF and/orPlGF from an hypoxic retina are thought to result in the development ofextraretinal neovascularization. Thus, inhibiting VEGF and/or PlGF wouldbe an effective way to prevent neovascularization.

A composition comprising a TPCD is useful to effect the liquefaction ofthe vitreous and/or the disinsertion or detachment of the vitreous fromthe retina and other tissues (e.g., epiretinal membranes, macula). As aresult of this vitreous liquefaction and/or vitreous detachment, thetractional forces of the vitreous on the retina and other tissues areminimized and the rate of natural turnover of fluids within the vitreousis accelerated. Accordingly, compositions comprising a TPCD areparticularly suitable for the treatment or prevention of many disordersof the eye, which benefit from vitreous liquefaction, posterior vitreousdetachment, decreasing extraretinal neovascularization and/oraccelerated clearance of toxins or other deleterious substances (e.g.,angiogenic factors, edema fluids, hemorrhagic blood etc.) from theposterior chamber of the eye and/or tissues adjacent to the posteriorchamber (e.g., retina or macula). Examples of such eye disordersinclude, but are not limited to, retinal detachment, retinal tear,vitreous hemorrhage, diabetic vitreous hemorrhage, proliferativediabetic retinopathy, non-proliferative diabetic retinopathy,age-related macular degeneration, macular holes, vitreomacular traction,macular pucker, macular exudates, cystoid macular edema, fibrindeposition, retinal vein occlusion, retinal artery occlusion, subretinalhemorrhage, amblyopia, endophthalmitis, retinopathy of prematurity,glaucoma and retinitis pigmentosa, and others in which the clinicalsymptoms of these disorders respond to TPCD administration. The presentinvention contemplates the treatment of disorders of the eye comprisingcontacting the vitreous with a composition comprising a TPCD. Suchcontact is expected to liquefy the vitreous and/or induce posteriorvitreous detachment and/or clear the vitreous cavity of blood or othertoxic substances and/or decrease extraretinal neovascularization,thereby treating or preventing the disorder.

The present invention is also directed to methods of preventing orinhibiting the onset of various disorders of the eye that are the resultof, or exacerbated by, vitreous adhesion to the retina and vitreouscontraction. In one embodiment, the methods of the present invention areable to prevent or inhibit the disorders, or complications resultingfrom a disorder in the eye of a subject without removing the vitreousfrom the eye. In particular, the invention is directed to a process oftreating a patient with proliferative disorders or at risk of developingproliferative disorders, such as, but not limited to, a diabeticpatient, by inducing posterior vitreous detachment as a prophylacticstep in preventing or delaying the onset of disorders associated withvitreous contraction or neovascularization into the vitreous. In oneembodiment of the invention, the composition is introduced into the eyeof a diabetic patient to inhibit progression of diabetic retinopathy.Preferably, the composition is introduced into the eye before theproliferative disorders occur. In one embodiment, the composition isintroduced into the vitreous of the eye before the onset ofproliferative disorders and allowed to remain in the eye indefinitelywithout removing the vitreous from the eye. In further embodiments, theinvention is directed to a process for inhibiting complications incentral and branch retinal vein occlusion, such as retinalneovascularization and macular edema by inducing posterior vitreousdetachment in a patient in need of such treatment. The present inventionprovides a process for treating impending or full-thickness macular hole(whether idiopathic or traumatic) by inducing posterior vitreousdetachment. Preventing or reducing the incidence of retinal detachment,retinal tears and retinal hemorrhage caused by vitreous contraction canbe achieved by inducing posterior vitreous detachment before suchdisorders occur and without removing the vitreous from the eye.

Many ophthalmic disorders have as a causative component, adestabilization of the blood-retina membrane. This destabilizationpermits various components (e.g., serum components, lipids, proteins) ofthe choriocapillaries to enter the vitreal chamber and damage theretinal surface. This destabilization is also a precursor to vascularinfiltration of the vitreal chamber, known as neovascularization.Neovascularization of the vitreous is dependent on the matrix of thevitreous. Thus, liquefaction of the vitreous, which removes the matrixin the form of the polymerized vitreous, blocks neovascularization. Inone embodiment, the invention provides a method of treating orpreventing eye disorders by preventing or reducing the incidence ofretinal neovascularization comprising contacting the vitreous with acomposition comprising a TPCD.

Several opthalmological disorders including diabetic retinopathy andtrauma result in the rupture or leakage of retinal blood vessels withresultant bleeding into the vitreous (i.e., vitreous hemorrhage).Vitreous hemorrhage typically manifests as clouding or opacification ofthe vitreous and is sometimes, but not always, accompanied by tearing ordetachment of the retina. In cases where the vitreous hemorrhage isaccompanied by a retinal tear or detachment, it is important that suchretinal tear or detachment be promptly diagnosed and surgicallyrepaired. Failure to promptly diagnose and repair the retinal tear ordetachment may allow photoreceptor cells of the retina, in the region ofthe tear or detachment, to become necrotic. Necrosis of thephotoreceptor cells of the retina may result in loss of vision.Furthermore, allowing the retinal detachment to remain unrepaired forsuch extended period of time may result in further vitreous hemorrhageand/or the formation of fibrous tissue at the site of the hemorrhage.Fibrous tissue may result in the formation of an undesirable permanentfibrous attachment between the vitreous body and the retina. In theabsence of any treatment, hemorrhagic clouding of the vitreous can takebetween 6-12 months or longer to clear sufficiently to allowtrans-vitreal viewing of the retina. In such cases, where a physicianwould need to repair any part of the retinal surface, or where aphysician would need to view the retinal surface of a patient that isprevented by an opaque or cloudy vitreous, a microsurgical procedureknown as vitrectomy may need to be performed. This procedure involvesremoval of all or a portion of the vitreous with a microsurgical cutterand the replacement of the vitreous with a clear liquid or othersubstance that allows the ocular cavity to maintain its shape. Standardvitrectomy surgical procedures are well known to those of ordinary skillin the art. In one embodiment, the present invention contemplatescontacting the vitreous with a composition comprising at least one TPCDas an adjunct to vitrectomy. In other embodiments, the vitreous iscontacted with the composition comprising at least one TPCD in theabsence of performing a vitrectomy.

The invention is illustrated further by the following examples, whichare not to be construed as limiting the invention in scope or spirit tothe specific procedures described therein. On the contrary, it is to beclearly understood that resort may be had to various other embodiments,modifications, and equivalents thereof which, after reading thedescription herein, may suggest themselves to those skilled in the artwithout departing from the spirit of the present invention and/or thescope of the appended claims.

Example 1 Vector Construction for Expression of Human Microplasminogenand Human Miniplasminogen in Pichia pastoris

(a) The pPICZα a Vector

The pPICZα A secretion vector purchased from Invitrogen Corporation(Carlsbad, Calif.) was used to direct expression and secretion ofrecombinant human microplasminogen and miniplasminogen in Pichiapastoris. Notable features of this vector include: (i) a 942 bp fragmentcontaining the alcohol oxidase 1 (AOX1) promoter that allowsmethanol-inducible, high level expression of recombinant protein inPichia, as well as targeted plasmid integration to the AOX1 chromosomallocus; (ii) the native transcription termination and polyadenylationsignal from the AOX1 gene; (iii) an expression cassette conferringzeocin resistance to Escherichia coli and Pichia pastoris; (iv) a ColE1origin of replication for propagation and maintenance of the plasmid inEscherichia coli; (v) a c-myc epitope and a polyhistidine (6×His) tag,which can be used for protein detection and purification; and (vi)unique restriction sites (for example, Sac I, Pme I, BstXI) that permitlinearization of the vector at the AOX1 locus for efficient integrationinto the Pichia genome.

In addition to the above features, this vector contains the secretionsignal of the Saccharomyces cerevisiae α-factor prepropeptide, allowingexpression of heterologous proteins as secreted proteins into themedium. The processing of the a factor mating signal sequence in pPICZαoccurs in two steps:

1. the preliminary cleavage of the signal sequence by the KEX2 geneproduct occurring between arginine and glutamine in the sequenceGlu-Lys-Arg*Glu-Ala-Glu-Ala, where * is the site of cleavage. However,the Glu-Ala repeats are not always necessary for cleavage by Kex2.2. the Glu-Ala repeats are further cleaved by the STE13 gene product. Insome cases where Ste13 cleavage is not efficient, the Glu-Ala repeatsare left on the NH₂-terminus of the expressed protein of interest.

Engineered immediately downstream of the α factor signal sequence in thepPICZα A vector is a multi-cloning site with recognition sequences forthe enzymes EcoR I, Sfi I, Kpn I, Xho I, Sac II and Xba Ito facilitatethe cloning of foreign genes. In addition to the Xho I site in themultiple cloning site, there is a Xho I recognition sequence at thecarboxyl-terminus of the a factor secretion signal, immediately upstreamof the Lys-Arg Kex2 cleavage site. This Xho I restriction site may beused to clone the gene of interest flush with the Kex2 cleavage site byusing a PCR cloning approach and an appropriate forward primer torebuild the sequence from the Xho I site to the arginine codon. Therecombinant protein of interest will then be expressed with a nativeNH₂-terminus.

(b) Expression Vector Construction for Microplasminogen

The nucleic acid sequence encoding the human microplasminogen protein(amino acids 543 to 791 (SEQ ID NO: 4)) was amplified (“PCR-rescue”)from the vector Fmyc-μPli (Lasters et al. in Eur. J. Biochem. 244:946,1997) using the Advantage cDNA polymerase mix available from Clontech(Palo Alto, Calif.). After a DNA template denaturation step of 3 minutesat 94° C., 30 rounds of thermal cycling were performed (30 seconds at94° C., 30 seconds at 50° C., 30 seconds at 72° C.), followed by a 2minutes final elongation step at 72° C. The following oligonucleotideprimers LY-MPLG1 (sense) and LY-MPLG2 (antisense) were used in thisreaction:

LY-MPLG1: 5′ (SEQ ID NO: 1)GGGGTATCT CTC GAG AAA AGA GCC CCT TCA TTT GAT TG LY-MPLG2: 5′(SEQ ID NO: 2) GTTTTTGT TCT AGA TTA ATT ATT TCT CAT CAC TCC CTC

The LY-MPLG1 primer had an annealing region corresponding to residues543-548 of human plasminogen (Ala-Pro-Ser-Phe-Asp-Cys) preceded by anon-annealing extension which included the last four residues of the αfactor mating signal (Leu-Glu-Lys Arg). In this extension, the Leu-Glucodons determine the Xho I restriction site (underlined) allowing thecloning of the gene of interest flush with the Kex2 cleavage site. TheLY-MPLG2 primer had an annealing region corresponding to the last sevenresidues of plasminogen, followed by a TAA stop-codon and anon-annealing region comprising a Xba I recognition sequence(underlined).

The amplified fragment having the expected size (˜780 bp) was digestedwith Xho I and Xba I, and directionally cloned into the vector pPICZα A.The recipient vector-fragment was prepared by Xho I and Xba Irestriction, and purified from agarose gel using the Qiaquick gelextraction kit (Qiagen GmbH, Germany). The E. coli strain TG1 (DSMZcollection #1208, Germany) was transformed with the ligation mixture,and zeocin resistant clones were selected. Based on restrictionanalysis, a plasmid clone containing an insert of the expected size wasretained for further characterization. Sequence determination of thevector pPICZα-MPLG1 (clone #5) using the primers 5′AOX and 3′AOX, whichwere provided in the EasySelect Pichia expression kit from InvitrogenCorporation (Carlsbad, Calif.), confirmed the precise insertion of themicroplasminogen coding region fused to the a factor mating signal, aswell as the absence of unwanted mutations in the coding region.

The determined nucleotide sequence and the deduced amino-acid sequenceof human microplasminogen used are represented in SEQ ID NO: 3 and SEQID NO: 4, respectively. Compared to the sequence previously determinedby Forsgren et al. in FEBS Lett. 213: 254, 1987, the nucleotide sequencediffers in 10 positions. However, the amino acid sequence was identical.

(c) Expression Vector Construction for Miniplasminogen

A pPICZα-derived secretion vector was constructed as follows forminiplasminogen expression, making use of the hereinabove describedpPICZα-MPLG1 vector.

A 500 bp DNA fragment encoding kringle five and part of the catalyticdomain of the miniplasminogen protein (amino acids 444 to 604 of SEQ IDNO:10) was amplified (“PCR-rescue”) from the vector FdTet-SN-miniPlg(Lasters et al., cited supra). After a DNA template denaturation step of3 minutes at 94° C., 30 rounds of thermal cycling were performed (10seconds at 94° C., 10 seconds at 50° C., 15 seconds at 72° C.), followedby a 2 minutes final elongation step at 72° C. The followingoligonucleotide primers LY-MINPLG1 (sense) and LY-MINPLG2 (antisense)were used in this reaction:

LY-MINPLG1: 5′ (SEQ ID NO: 5)GGGGTATCT CTC GAG AAA AGA GCA CCT CCG CCT GTT GTC CTG CTT CCLY-MINPLG2: 5′ (SEQ ID NO: 6) GCA GTG GGC TGC AGT CAA CAC CCA CTC

The LY-MINPLG1 primer has an annealing region corresponding to residues444-452 of plasminogen (Ala-Pro-Pro-Pro-Val-Val-Leu-Leu-Pro) preceded bya non-annealing extension which included the last four residues of thefactor mating signal (Leu-Glu-Lys-Arg). In this extension, the Leu-Glucodons determine the Xho I restriction site (underlined) allowing thecloning of the gene of interest flush with the Kex2 cleavage site.

The LY-MINPLG2 primer has an annealing region corresponding to theresidues 596-604 of human plasminogen(Glu-Trp-Val-Leu-Thr-Ala-Ala-His-Cys). This annealing region of thecatalytic domain, also present in the microplasminogen expressionvector, comprises a unique Pst I recognition sequence (underlined).

The amplified fragment having the expected size was digested with Xho Iand Pst I, and directionally cloned into the recipient vector fragmentderived from pPICZα-MPLG1 (described above). The recipientvector-fragment was prepared by Xho I and Pst I restriction, andpurified from agarose gel using the Qiaquick gel extraction kit (QiagenGmbH, Germany). The E. coli strain TG1 (DSMZ collection #1208, Germany)was transformed with the ligation mixture, and zeocin resistant cloneswere selected. Based on restriction analysis, a plasmid clone containingan insert of the expected size was retained for furthercharacterization. Sequence determination of the vector pPICZα-KMPLG1(clone #3), using the primers 5′AOX and 3′AOX, confirmed the preciseinsertion of the amplified fragment fused to the α-factor mating signal,as well as the absence of any mutations in the cloned region.

Example 2 Method of Preparing Recombinant, Stabilized Microplasmin

(a) Transformation of Pichia with pPICZα-MPLG1

Ten μg of the vector pPICZα-MPLG1 was digested with Pme I, whichlinearizes the vector in the 5′ AOX1 region. The DNA was precipitatedand concentrated to about 0.33 μg/μl in sterile distilled water, and 5μl was used to transform competent Pichia pastoris X33 cells preparedaccording to the manual provided in the EasySelect Pichia expressionkit.

(b) Selection of a High-Expression Strain

The selection of a high-expression strain was performed as follows.Zeocin resistant transformants were selected on YPDSZ plates (1% yeastextract, 2% peptone, 2% glucose, 1M sorbitol, 2% agar, 100 μg/mlzeocin). Thirty-four single colonies were inoculated in 10 mlBMYZ-glycerol medium (1% yeast extract, 2% peptone, 1% glycerol, 100 mMpotassium phosphate, pH 6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin,100 μg/ml zeocin) in 50 ml Falcon tubes and cultured for 16 hours at 30°C. The cells were pelleted and re-suspended in 2 ml of BMYZ-methanolmedium (same as BMYZ-glycerol but with 0.5% methanol instead ofglycerol) to induce expression from the AOX1 promoter, and cultured for40 hours. 4 pulses of 0.5% methanol were supplied to the cultures overthis period (after 6, 22, 26 and 30 hours). At the end of the inductionculture, the presence of microplasminogen in the culture supernatant wasestimated as described by Lijnen et al. in Eur. J. Biochem. 120:149,1981. Briefly, the microplasminogen in pure or 10-fold dilutedsupernatants was incubated with urokinase for 30 minutes to activatemicroplasminogen to microplasmin. The generated microplasmin activity,as determined by its amidolytic activity measured with the chromogenicsubstrate S2403 (available from Chromogenix, Antwerp, Belgium) atdifferent times, was compared to the activity of known amounts ofpurified plasmin or microplasmin preparations. The clone X33-MPLG1 #5,showing the highest microplasmin activity after urokinase activation,was selected for subsequent large scale production. This clone wasdeposited under the provisions of the Budapest Treaty with the BelgianCoordinated Collections of Microorganisms (BCCM-MUCL-COLLECTION) on Dec.12, 2001, under Accession Number MUCL 43676.

(c) Fermentation

Fermentation of X33-MPLG1#5 at a 50 liter scale was carried out in foursteps as follows. Two liter flask cell cultures were performed for 23hours at 30° C. in 400 ml YSG+ (6 g/l of yeast extract, 5 g/l of soyapeptone, 20 g/l of glycerol) using an inoculum of 0.7 ml (of cell bankvial number glycerol OOC17) and 270 rpm agitation, yielding (at the endof the pre-culture step) an OD600 of 15. Fermentation was then performedin a MRP80 fermentation device in 30 l basal medium (26.7 ml/l H3PO485%, 1.05 g/l CaSO4.2H2O, 18.2 g/l K2SO4, 14.9 g/l MgSO4.7H2O, 4.13 g/lKOH, 40 g/l of 100% glycerol and 4.76 ml/l PTM1 salt solution[comprising 6 g/l CuSO4.5H2O, 0.08 g/l NaI, 3.36 g/l MnSO4.H2O, 0.2 g/lNaMoO4.2H2O, 0.02 g/l Boric acid, 0.82 g/l CoCl2.6H2O, 20 g/l ZnCl2, 65g/l FeSO4.7H2O, 0.2 g/l d-biotin and 5 ml/l HSSO4]), using 600 mlinoculum at 30° C. with an air flow of 50 l/min at atmospheric pressure,dissolved oxygen (DO)>20% and 200-500 rpm agitation, pH being maintainedat 5.8 with 12.5% ammonia. At 24 hours and OD 600 of 50 (end of batchstep), glycerol depletion was evidenced by a rapid increase of dissolvedoxygen. Glycerol feeding (632 g/l glycerol 100% and 12 ml/l PTM1)increased the OD 600 up to 258 in 24 hours. Methanol feeding was thencarried out with an increasing flow of up to 250 ml/h within 6 hours,which was maintained for 66 hours using 988 ml/l methanol and 12 ml/lPTM1 to reach an OD 600 of 352 at the end of culture. Fermentation ofX33-MPLG1#5 at a 350 liter scale provided proportionally similarresults.

(d) Purification

The harvest was then purified in a three-steps process comprising cationexchange expanded bed chromatography, hydrophobic chromatography andaffinity chromatography as follows:

i) Cation Exchange Expanded Bed Chromatography

Cation exchange expanded bed adsorption chromatography was conductedwith Streamline SP (available from Pharmacia Biotechnology, Cat. No.17-0993-01/02) packed in a Streamline 200 column (PharmaciaBiotechnology Cat No. 18-1100-22) with a bed volume of 5,120 cm³,expanded and equilibrated by applying an upward flow of 1 M NaCl, 25 mMsodium acetate (CH₃COONa.3 H₂O) buffer, pH 6.0, for two column volumesfollowed by column volumes of 25 mM sodium acetate buffer, pH 6.0. Thefermentation broth was on line diluted (7×) with water and passedupwards through the expanded bed at a flow rate of 1000 ml/min. Looselybound material was washed out with the upward flow of 25 mM sodiumacetate buffer pH 6.0. The column adaptor was then lowered to thesurface of the sedimented bed at a height of 16.3 cm. Flow was reversedand the captured proteins eluted with 2 column volumes of 0.5 M NaCl, 25mM sodium acetate buffer, pH 6.0. Solid ammonium sulfate was added tothe eluted Streamline fraction to reach 30% saturation (164 g ammoniumsulfate per liter of eluted Streamline fraction) and the mixture wasgently stirred at 4-8° C. for 1 hour.

ii) Hydrophobic Chromatography

Hydrophobic chromatography was conducted with Hexyl TSK 650C (availablefrom Toso-Haas Cat. No. 19027) packed in a Vantage 180/500 column(available from Millipore, Cat. No. 87018001) with a packed volume of2,700 cm³ at 4-8° C. The eluted streamline fraction was loaded on thecolumn at a flow rate of 38 l/hour. The column was then washed with 1.5column volumes of 25 mM sodium acetate buffer, pH 6.0, containing 164g/l ammonium sulfate and eluted from the column with 7 column volumes of25 mM sodium acetate buffer, pH 6.0.

iii) Affinity Chromatography

Affinity chromatography was conducted with Blue Sepharose 6 Fast Flow(available from Pharmacia Biotechnology, Cat. No. 17-0948-02/03) packedin a Vantage 130/500 column (available from Millipore, Cat. No.87013001) with a packed volume of 3,186 cm3 at 4-8° C. The elutedfraction was loaded on the column at a flow rate of 20 l/hour, andwashed with one column volume of 25 mM disodium hydrogenophosphate(Na₂HPO₄.12 H₂O) buffer, pH 7.0. The microplasminogen protein fractionwas eluted from the column with 5 column volumes of 0.5 M NaCl, 25 mMdi-sodium hydrogenophosphate buffer, pH 7.0, and kept frozen at −20° C.The purity of the material was above 98% as demonstrated by SDS gelelectrophoresis.

(e) Quantitative Activation to and Stabilization of Microplasmin i)Quantitative Activation

The activation of microplasminogen to microplasmin was performed at 23°C. for 30 minutes at a molar ratio of 0.5% of a staphylokinase variantSY162 in 0.5 M NaCl, 25 mM di-sodium hydrogenophosphate (Na₂HPO₄.12 H₂O)buffer, pH 7.0. SY162 is a staphylokinase variant with reducedimmunogenicity comprising 12 amino-acid substitutions (K35A, E65Q, K74R,E80A, D82A, T90A, E99D, T101S, E108A, K109A, K130T and K135R) ascompared to wild-type, as described by WO 99/40198. Solid ammoniumsulfate was added to microplasmin at a final concentration of 1 M (132g/l) and the mixture stirred at 4-8° C. for 15 minutes.

ii) Hydrophobic Chromatography

Hydrophobic chromatography was conducted with Phenyl Sepharose 6 FastFlow (available from Pharmacia Biotechnology, Cat. No. 17-0965-03/05)packed in a BPG 100/500 column (available from Pharmacia Biotechnology,Cat. No. 18-1103-01) having a packed volume of 1,738 cm³, equilibratedwith 4 column volumes of 25 mM Na₂HPO₄.12 H₂O buffer, pH 7.0, containing0.1 M of the stabilizing agent, tranexamic acid (available fromBournonville Pharma, Braine-L'Alleud, Belgium) and 1 M (NH₄)₂SO₄ pH 7.0,at 4-8° C. The activated product was loaded on the column at a linearflow rate of 18 l/hour and washed with 4.5 column volumes of 25 mMNa₂HPO₄.12 H₂O buffer, pH 7.0, containing 0.1 M tranexamic acid and 1 M(NH₄)₂SO₄. Microplasmin was eluted from the column at a linear flow rateof 6 l/hour with 5 column volumes of 25 mM Na₂HPO₄.12 H₂O buffer, pH7.0, containing 0.1 M tranexamic acid and 0.7 M (NH₄)₂SO₄ andequilibrated with phosphate buffered saline containing 0.1 M tranexamicacid. Staphylokinase variant SY162 was eluted from the column with 25 mMNa₂HPO₄.12H₂O buffer, pH 7.0 containing 0.1 M tranexamic acid. Thisprocedure removed above 99% of staphylokinase from the microplasmin peakas demonstrated with a specific ELISA assay.

iii) Concentration and Diafiltration by Tangential Ultrafiltration

In this step, the eluate from step (ii) was concentrated and the bufferwas exchanged for the low pH citric acid buffer. The tranexamic acid ofstep (ii) was removed during this step and microplasmin was stabilizedby the low pH citric acid buffer.

Ultrafiltration was conducted with 2 Pellicon 2 Biomax membranes (5 kDa,2.5 μm, available from Millipore, Bedford, Mass., Cat. No. P2B005A25) at2-8° C. The membranes were mounted in a Pellicon 2 Process Holderconnected to a Microgon Pump Cart System (available from Microgon,Laguna Hills, Calif.). The membranes were washed with purified water andmembrane integrity tested before operation. Sanitization was performedby continuous recirculation with 0.5 M NaOH for 60 minutes and with 0.1M NaOH during 60 minutes. The sanitization step deep cleans the membraneto eliminate any potential trace of protein left on the membrane beforeapplying the sample. The membranes were then rinsed with 5 mM citricacid, pH 3.1, until the permeate reached a pH of 3.1. The pH of thePhenyl Sepharose eluate was adjusted to 3.1 and the protein wasconcentrated to 4 mg/ml by ultrafiltration. Diafiltration was performedfor 60 to 90 minutes against 5 volumes of 5 mM citric acid, pH 3.1.Yields (expressed in grams) of three runs performed on a 50 literfermentation apparatus are summarized in Table 2.

TABLE 2 Run 1 Run 2 Run 3 Fermentor 220 240 ND Streamline 50 79 130 Hexyl 36 37 ND Blue 25 28 30 Phenyl 17 20 26 Diafiltration — — 22 (Key:ND: not determined)

iv) Sterile Filtration (0.2 μm)

This step was performed to ensure absence of microbial contamination.

Mannitol was added at 2-8° C. to a concentration of 1.5 g/g of proteinand sterile filtration performed at 23° C. on a Millipak 100 filter(size 500 cm²) (available from Millipore, Cat. No. MPGL10CA3) and rinsedwith about 500 ml of 5 mM citric acid, pH 3.1, with a peristaltic pumpat a flow rate of 500 ml/minute. The filtrate was collected in a sterileand pyrogen free bag and stored at −20° C.

Example 3 Method of Preparing a Recombinant, Stabilized Miniplasmin

About 15 μg of the vector pPICZα-KMPLG1 was digested in a 20 μl reactionwith Pme I, which linearizes the vector in the 5′ AOX1 region. Thelinear DNA (3 μg) was used to transform competent Pichia pastoris X33cells prepared according to the manual provided in the EasySelect PichiaExpression kit.

The selection of high-expression strain was performed essentially asfollows. Zeocin resistant transformants were selected on YPDSZ plates(as defined in example 2). Fifty isolated colonies were inoculated in 15ml BMYZ-glycerol medium (as defined in example 2) in 50 ml Falcon tubesand cultured for 16 hours at 30° C. The cells were pelleted andre-suspended in 1.5 ml of BMYZ-methanol medium (as defined in example 2)to induce expression from the AOX1 promoter, and cultured for 40 hours.3 or 4 pulses of 0.5% methanol were regularly supplied to the culturesover this period. At the end of the induction culture, the presence ofminiplasminogen in the culture supernatant was estimated as described byLijnen et al. (cited supra). Briefly, the miniplasminogen in 10-folddiluted supernatants was incubated with streptokinase for 10 minutes toform an active complex. The generated miniplasmin activity, asdetermined with the chromogenic substrate S2403 (see example 2) atdifferent times, was compared to the activity of known amounts of apurified plasminogen preparation. In these conditions, all tested clonesproduced miniplasminogen with yields varying between 3 and 15 mg/l. Thetwo clones X33-KMPLG1 #6 and X33-KMPLG1 #25, showing the highestminiplasmin activity, were selected for subsequent large scaleproduction. These two clones were deposited under the provisions of theBudapest Treaty with the Belgian Coordinated Collections ofMicroorganisms (BCCM-MUCL-COLLECTION) on Dec. 4, 2003 and have beenaccorded Accession Number MUCL 45309 (clone X33-KMPLG1 #6) and AccessionNumber MUCL 45308 (clone X33-KMPLG1 #25).

Example 4 Novel Method of Fixation

This experiment was carried out to establish a fixation technique thatis reliable to investigate the effects of media and different agents onposterior vitreous detachment (PVD) in normal porcine eyes.

Freshly isolated porcine eyes obtained from the slaughterhouse wereeither immediately processed or allowed to sit at room temperature forup to 6 hours. The cornea was removed to facilitate fixation. Eyes werefixed in Peter's solution (1.25% glutaraldehyde/1% paraformaldehyde in0.08 M cacodylate buffer pH 7.4) at 0° C. for 24 to 36 hours to stopenzymatic reactions. The eyes were then washed with 0.1 M cacodylatebuffer pH 7.4 and progressively dehydrated in progressively higherconcentrations of ethanol up to 100%.

Both the freshly processed eyes and the eyes allowed to sit for 6 hoursshowed no significant change in the ultrastructure of the retina, andthe vitreous remained attached to the retinal surface. Thus, this methodprovides a non-traumatic fixation procedure for eye tissue and minimizesthe possibility of the separation of the vitreous from the retinalsurface. Furthermore, this procedure allows the whole retinal surface,from the optic nerve to the retinal periphery to be studied.

Example 5 Effect of Microplasmin on the Vitreoretinal Interface inPost-Mortem Porcine Eyes

This experiment was carried out to determine the ability of microplasminto disinsert the posterior vitreous cortex from the inner limitingmembrane of the retinal surface.

Microplasmin was used at the following doses: 0.0625, 0.125, 0.156, 0.25and 0.390 mg, in a volume of 0.1 ml of the intraocular irrigatingsolution, BSS PLUS®. The pH of these microplasmin solutions ranged from7.92 for the 0.0625 mg dose to 6.52 for the 0.390 mg dose.

The microplasmin solutions disclosed above were separately injected intothe vitreous humor of eyes obtained from freshly slaughtered pigs atroom temperature (24° C.). The eyes were fixed as described in Example4, after 15 minutes, 30 minutes, 60 minutes or 120 minutes followinginjection of microplasmin. Posterior vitreous detachment (PVD) wasobserved after 1 hour of treatment with 0.0625 mg of microplasmin,whereas PVD was visible from about 30 minutes after the injection forall doses including and above 0.125 mg of microplasmin. This detachmentwas most apparent around 120 minutes post-injection (FIG. 4, Panel A),in all sections of the retina surface, except near the vitreous base(the zone extending from the peripheral retina to the ora serrata wherethe adhesion to the vitreous is strong). In addition to posteriorvitreous detachment (FIG. 4, Panels B-E), electron microscopy showedthat the structure of the vitreous was altered to have less fibrillarystructure present. The fibrillar structure was modified to a moreamorphous, ground glass consistency, which indicates liquefaction of thevitreous humor (FIG. 4, Panel F).

Doses lower than 0.25 mg did not result in any ocular or retinaltoxicity as determined by gross examination or histopathology, includingelectron microscopy. In particular, there was no sign of autolysis.Vacuolation of cells is often seen as an early sign of autolysis and atdoses lower than 0.390 mg, no vacuolation was observed. We also examinedother ocular structures by electron microscopy (FIG. 5, Panels A and B).No structural alterations to the retina were observed at doses lowerthan 0.390 mg. However, in a few eyes treated with 0.25 mg ofmicroplasmin, retinal elevations and small numbers of inflammatory cellswere sparsely distributed on the retinal surface. Gross histology of theeye treated with the highest dose of microplasmin (0.390 mg), indicatedthat the retinal interface had a whitish appearance. Electron microscopyof this eye revealed that there were multiple small elevations in theretinal surface, which suggests localized retinal detachment.

These experiments show that microplasmin used at a dosage of 0.06 to 0.2mg resulted in consistent separation of the posterior hyaloid withoutinducing any ultrastructural changes in the retina. The posteriorhyaloid separation is not only at the optic nerve but also all the wayto the vitreous base. The posterior hyaloid separation leaves a clear,smooth retinal surface on which no collagen fibers can be recognizedusing high-electron microscopic scanning (12,000× magnification), amagnification that is high enough to exclude the possibility ofundetected fibers.

Example 6 Posterior Vitreous Detachment in Human Post-Mortem Eyes

This experiment was performed to determine whether microplasmin couldefficiently induce vitreoretinal separation in human eyes.

Methods (a) Dosage and Treatment of Human Post-Mortem Eyes

Twenty-six human globes with no known eye pathology were obtained fromthe Munich eye bank. These globes were removed from 13 donors, whoseages ranged from 34 to 69 years, within 19 hours of their death. Afterharvesting the cornea using a 14 mm diameter trephine, the 26 globeswere incubated in a moist chamber at 37° C. for 15 minutes. 0.2 ml ofmicroplasmin was then injected into the vitreous cavity of thirteeneyes. Specifically, 1.25 mg of microplasmin was diluted with 4 ml, 2 ml,or 1.5 ml of the intraocular irrigating solution, BSS PLUS® to achieveconcentrations of 0.3125 mg/ml, 0.625 mg/ml, and 0.9375 mg/mlrespectively. A total volume of 0.2 ml of these solutions was injectedinto the vitreous cavity, resulting in a final dose of 62.5 μg, 125 μg,and 188 μg of microplasmin respectively within the eye. The 13 felloweyes, which served as controls, received an injection of 0.2 ml ofbalanced salt solution (BSS PLUS®).

Of the 13 eyes treated with microplasmin, 9 eyes were treated by anintravitreal injection of microplasmin alone. A dose of 62.5 μg ofmicroplasmin (pH 7.4) was injected into the vitreous cavity of two eyes;a dose of 125 μg of microplasmin (pH 7.2) was injected into the vitreouscavity of 5 eyes; and a dose of 188 μg of microplasmin (pH 7.2) wasadministered in 2 eyes. Of the remaining 4 eyes, two were treated with62.5 μg of microplasmin and 0.6 ml of sulfurhexafluoride (SF₆) and twowere treated with 125 μg of microplasmin and 0.6 ml ofsulfurhexafluoride (SF₆). The additional treatment with SF₆ wasperformed because of previous reports that plasmin only induces PVD incombination with vitrectomy or gas injection. The dosing and treatmentdiscussed above are summarized in Table 3.

TABLE 3 Dose of microplasmin Number of eyes in μg Treatment 2 62.5Intravitreal injection 5 125 Intravitreal injection 2 188 Intravitrealinjection 2 62.5 Intravitreal injection and gas tamponade (0.6 ml SF₆) 2125 Intravitreal injection and gas tamponade (0.6 ml SF₆)

Following treatment, all eyes were incubated at 37° C. for 30 minutes.After that time, the globes were placed in 4% paraformaldehyde, and 0.1ml of fixative (4% paraformaldehyde) was also injected into the vitreouscavity to stop enzymatic action within the eye. The globes that weretreated with microplasmin and SF₆ were fixed with the posterior pole inan upright position.

(b) Scanning and Transmission Electron Microscopy

The globes were then hemisected along the pars plana, and the anteriorsegment was discarded. A corneal trephine of 12.5 mm diameter was slowlymoved through the vitreous and the posterior pole was punched out.Retinal specimens for scanning and transmission electron microscopy werethen obtained from the posterior pole using a corneal trephine of 4 mmdiameter.

Retinal discs for scanning electron microscopy were post-fixed in 2%osmium tetroxide (Dalton's fixative), dehydrated in ethanol, dried tothe critical point, sputter-coated in gold, and photographed using aISM-35 CF electron microscope (JEOL®, Tokyo, Japan).

Specimens for transmission electron microscopy were post-fixed inDalton's fixative, dehydrated, and embedded in EPON™. Semithin sectionswere stained with 2% toluidine blue. Ultrathin sections were contrastedwith uranyl acetate and lead citrate, and analyzed using a Zeiss EM 9electron microscope (Zeiss, Jena, Germany).

Two observers independently evaluated the electron micrographs. Eachobserver evaluated the degree of vitreoretinal separation by decidingwhether a continuous or discontinuous network of collagen fibrilscovered the inner limiting membrane (ILM), or whether single or sparsecollagen fibrils were present at the ILM, or whether the ILM was devoidof any collagen fibrils (bare ILM).

Results (a) Scanning Electron Microscopy

Scanning electron microscopy (SEM) of post-mortem human eyes injectedwith 62.5 μg of microplasmin revealed a posterior vitreous detachmentleaving a discontinuous network of collagen fibrils covering the ILM(FIG. 6, Panel A). SEM of eyes injected with 125 μg (FIG. 6, Panel B)and 188 μg (FIG. 6, Panel C) of microplasmin respectively revealed abare ILM consistent with complete vitreoretinal separation. Both thesehigher doses resulted in a similar ultrastructure of the vitreoretinalinterface.

In eyes injected with 62.5 μg of microplasmin and SF₆, remnants ofcortical vitreous covering the ILM were observed (FIG. 6, Panel D).However in eyes injected with 125 μg of microplasmin followed by gastamponade, complete vitreoretinal separation consistent with a bare ILMwas observed (FIG. 6, Panel E).

In contrast to the microplasmin-treated eyes discussed above, controleyes did not exhibit posterior vitreous detachment as determined by SEM(FIG. 6, Panel F). These results are summarized in Table 4.

TABLE 4 Degree of residual Dose of cortical vitreous microplasmin in μgTreatment Treated eyes Control eyes 62.5 Intravitreal injection ++ +++125 Intravitreal injection − +++ 188 Intravitreal injection − +++ 62.5Intravitreal injection ++ +++ and gas tamponade 125 Intravitrealinjection − +++ and gas tamponade [Key: +++ continuous network ofcollagen fibrils; ++ discontinuous network of collagen fibrils; + sparsecollagen fibrils; − no collagen fibrils, bare ILM]

(b) Transmission Electron Microscopy

The intraretinal morphology of all microplasmin-treated eyes wasunchanged compared to control eyes. The ultrastructure of the ILM waswell preserved in microplasmin-treated eyes (FIG. 7, Panel A), ascompared to the control eye (FIG. 7, Panel B).

Conclusion

These data indicate that intravitreal injection of microplasmin caninduce a cleavage between the vitreous cortex and the inner limitingmembrane of the human eye without vitrectomy or any other surgicalintervention. 125 μg of microplasmin cleaves the human vitreoretinaljunction within 30 minutes. In terms of enzymatic action, 125 μg ofmicroplasmin is the equivalent dose of 2 U of plasmin (Sigma-Aldrich,Munich, Germany), which caused complete vitreoretinal separation in pigcadaver eyes and in human donor eyes. These data also show that theapplication of a gas bubble into the vitreous of a microplasmin-treatedeye did not affect the dose needed to cleave the vitreoretinal junction.

Example 7 In Vivo Analysis of Microplasmin-Induced Posterior VitreousDetachment

The purpose of these experiments was to determine the utility ofmicroplasmin for PVD in vivo.

Methods (a) The Feline Model

The feline retina has been extensively studied by anatomists andphysiologists making this a useful model to assess the safety ofpharmacologically induced PVD. Like the human retina, the cat retina isrod-dominated and has an intraretinal circulation, outside the fovea.This is in contrast to the rabbit retina, which has no intraretinalvessels. The rabbit inner retina is perfused by vasculature that lies onits vitreal surface, and this limits the value of experimental studiesprimarily focused on the vitreoretinal interface in the rabbit. Foryears, the feline model has provided high-quality data on the cellularresponses of the retina to detachment. Thus, we used a feline model tostudy the utility of microplasmin in effecting PVD in vivo.

Five adult domestic cats aged between 12 and 23 months were anesthetizedwith an intramuscular injection of 0.5 ml of ketamine (Ketaset,Park-Davis, Eastleigh, UK) and 0.3 ml of medetomide (Dormitor, Pfizer,UK). The anesthetized cats received an intravitreal injection of 14.5 μgor 25 μg of microplasmin, while the fellow eyes of these cats receivedan injection of balanced salt solution (BSS-PLUS®) and served ascontrols. Of the 5 cats used in this study, 3 cats received anintravitreal injection of 25 μg of microplasmin. One of these cats wassacrificed after one day of receiving the injection; the second wassacrificed after 3 days and the third was sacrificed after 3 weeks. Thetwo remaining cats were injected with 14.5 μg microplasmin. Of these,one was sacrificed after 3 days while the other was sacrificed after 3weeks.

(b) Scanning and Transmission Electron Microscopy

The globes were removed from the sacrificed cats, fixed and processedfor electron microscopy as described for human post-mortem eyes inExample 6. Electron micrographs were evaluated independently by twoobservers. Each observer evaluated the degree of vitreoretinalseparation by deciding whether a continuous or discontinuous network ofcollagen fibrils covered the ILM, or whether single or sparse collagenfibrils were present at the ILM, or whether the ILM was devoid of anycollagen fibrils.

(c) Confocal Microscopy

The eye specimens were rinsed in phosphate buffered saline (PBS) andorientated in 5% agarose (Sigma, St Louis Mo., USA) prepared in PBS. Onehundred micrometer thick sections were cut using a vibratome (TechnicalProducts International, Polysciences, Warrington, Pa., USA) andincubated in normal donkey serum (1:20; Dianova, Hamburg, Germany) inPBS containing 0.5% bovine serum albumin (BSA; Fisher Scientific,Pittsburgh, Pa., USA), 0.1% Triton X-100 (Roche Boehringer, Mannheim,Germany) and 0.1% sodium azide (Sigma-Aldrich, Munich, Germany) (thisPBS solution containing BSA, Triton and azide is referred to as PBTA)overnight at 4° C. on a rotator. After removal of blocking serum,primary antibodies were added in six sets of pairs: anti-glialfibrillary acidic protein (GFAP; 1:500; DAKO, Hamburg, Germany) withanti-collagen IV (1:50; DAKO); anti-vimentin (1:50; DAKO) withanti-fibronectin (1:400; DAKO); anti-synaptophysin (1:50; DAKO) withanti-neurofilament (1:25; DAKO); anti-laminin (1:25; DAKO) with anti-CD68 (1:50; DAKO); anti-red/green opsin (1:100; Santa Cruz Biotechnology,USA) with anti-rhodopsin (1:200; Santa Cruz Biotech); anti-blue opsin(1:100; Santa Cruz Biotech, Santa Cruz, Calif., USA) with anti-rhodopsin(1:200; Santa Cruz Biotech). The specifity of the antibodies used inthis experiment is listed in Table 5.

TABLE 5 Antibody Specifity Anti-glial fibrillic acidic proteinIntermediate filament proteins of (GFAP) Anti-vimentin Muller cellsAnti-neurofilament Neurofilaments in ganglion cells and in horizontalcells Anti-synaptophysin Synaptic vesicles in plexiform layersAnti-red/green opsin Cones Anti-blue opsin Anti-rhodopsin RodsAnti-fibronectin Fibronectin Anti-laminin Laminin Anti-collagen IVCollagen type IV Anti-CD68 Macrophages

After overnight incubation at 4° C. on a rotator, sections were rinsedin PBTA and incubated again overnight at 4° C. with the secondaryantibody. Donkey anti-mouse and donkey anti-rabbit secondary antibodieswere used for each combination of primary antibodies, conjugated to Cy2or Cy3 (Dianova, Hamburg, Germany). All secondary antibodies were usedat a dilution of 1:100, and all the antibodies were diluted in PBTA. Thesections were then rinsed, mounted in N-propyl gallate in glycerol andviewed on a laser scanning confocal microscope (LSM 510, Zeiss,Germany).

Results (a) Scanning Electron Microscopy

One day following intravitreal injection of 25 μg microplasmin, sparsecollagen fibrils covered the ILM (FIG. 8, Panel A). Three days aftertreatment, 25 μg microplasmin resulted in complete vitreoretinalseparation (FIG. 8, Panel B); there were no remnants of collagen fibrilsleft on the vitreoretinal interface. The eye which received 14.5 μg ofmicroplasmin revealed sparse collagen fibrils covering the ILM 3 daysafter the injection (FIG. 8, Panel C). 21 days after treatment, with14.5 μg (FIG. 8, Panel D) and 25 μg of microplasmin (FIG. 8, Panel E) abare ILM was observed. All fellow control eyes had a dense network ofcollagen fibrils covering the retina (FIG. 8, Panel F). These data aresummarized in Table 6.

TABLE 6 Degree of residual Dose of Duration of cortical vitreousmicroplasmin in μg treatment in days Treated eyes Control eyes 14.5 3 ++++ 14.5 21 − +++ 25 1 + +++ 25 3 − +++ 25 21 − +++ [Key: +++ continuousnetwork of collagen fibrils; ++ discontinuous network of collagenfibrils; + sparse collagen fibrils; − no collagen fibrils, bare ILM]

(b) Light and Transmission Electron Microscopy

Regarding the cytoarchitecture of the retina, no difference was observedbetween microplasmin-treated eyes (FIG. 9, Panel A) and control eyes(FIG. 9, Panel B). The ultrastructure of the inner retina and the ILM ofmicroplasmin treated eyes (FIG. 9, Panels C and E) were well preservedcompared to the inner retina and the ILM of control eyes (FIG. 9, PanelsD and F).

(c) Laser Confocal Microscopy

In microplasmin-treated eyes and in control eyes, the end-foot portionof Müller cells was clearly labeled by anti-GFAP (FIG. 10, Panels A andB) and anti-vimentin (FIG. 10, Panels C and D). There was no extendedstaining of Müller cell processes beyond the inner nuclear layer. Nosignificant staining was observed with anti-collagen IV andanti-fibronectin (data not shown). This may relate to species specifityof these antibodies. The ILM was stained by anti-laminin. Fewmacrophages were present in both treated eyes and control eyes. Theganglion cell axons and dendrites, the horizontal cells, and the innerand outer plexiform layer were clearly labeled by anti-neurofilament andanti-synaptophysin (FIG. 10, Panels E and F). The photoreceptor layerwas labeled by anti-neurofilament. There was no difference betweenmicroplasmin-treated eyes (Panels A, C, E) and control eyes (FIG. 10,Panels B, D and F) at any time point of the study, with respect to anyof the antibodies used.

Discussion

To assess the cleaving effect of microplasmin at the vitreoretinalinterface in vivo, we administered two different doses into the vitreouscavity of five adult cats. The first dose we used was 25 μg ofmicroplasmin, which is one-fifth of the dose that was found sufficientto induce complete PVD in human post-mortem eyes. Notably 25 μg ofmicroplasmin is equivalent to 0.4 U of plasmin (Sigma), and clinically0.4 U of autologous plasmin has been applied to the vitreous cavity ofhuman eyes with macular holes and diabetic retinopathy. The second doseof 14.5 μg of microplasmin is equivalent to a dose of 25 μg ofmicroplasmin in the human eye, if one adjusts for the smaller vitreousvolume of the cat eye (roughly 60% of the vitreous volume of the humaneye).

Three days following an intravitreal application of 25 μg ofmicroplasmin in the cat eye, there was complete vitreoretinalseparation, whereas one day after treatment some collagen fibrils werestill present at the vitreoretinal interface. This indicates that theeffect of microplasmin continues beyond 24 hours and stands in strikingcontrast to the rapid inactivation of plasmin in the blood by itsnatural antagonist alpha-2-antiplasmin. One reason for the longevity ofmicroplasmin activity may be that alpha-2-antiplasmin is saturated byanother substrate, or that microplasmin has a different affinity for theantiplasmin antagonist compared to plasmin. Another possible reasoncould be that pathways downstream of microplasmin (for example,activation of collagenases or matrix metalloproteinases) remain activeafter microplasmin is inactivated by alpha-2-antiplasmin.

The cytoarchitecture of the retina of microplasmin-treated eyes wasunchanged compared to control eyes. In ultrastructural terms, there wasno difference in the retinal anatomy between microplasmin-treated eyesand control eyes. The ILM and the retina were well preserved in allspecimens. Additionally we did not observe any signs of an inflammatoryreaction following microplasmin injection. Specifically, electronmicroscopy and laser confocal microscopy did not show any evidence ofinflammatory cellular infiltration of the retina.

In the feline model, retinal detachment produces a significantproliferation of Müller cells and a massive upregulation of intermediatefilament proteins in their cytoplasm, such as glial fibrillic acidicprotein (GFAP) and vimentin. This Müller cell response is widely knownas gliosis, and is supposed to play a key role in the complex cellularresponses of the retina to detachment. In the normal retina, Müllercells appear quiescent and express very small amounts of GFAP andvimentin. However, even vitrectomy without inducing retinal detachmenthas been shown to cause upregulation of GFAP. Recent work by our groupdemonstrated marked upregulation of intermediate filament proteinsfollowing attempted peeling of the ILM in cat eyes (unpublished data).These data point to the high reactivity of Müller cells to any form ofsurgical trauma.

In the present study, we did not observe any change of Müller cellreactivity following induction of PVD by microplasmin. Moreover, therewas no difference between treated eyes and control eyes with respect toany antibody applied. The quiescent state of Müller cells at any timepoint of the study in association with the unchanged ultrastructure andimmunoreactivity of the retina provides experimental evidence pointingto the safety of microplasmin in inducing PVD.

In conclusion, these studies show that microplasmin is effective ininducing PVD in vivo. In addition, these studies indicate thatmicroplasmin appears safe in that no retinal alterations were observedat the ultrastructural level.

Example 8 Evaluation of the Effects of Microplasmin on Porcine Vitreousby Use of Dynamic Light Scattering

This study was conducted to evaluate the effects of microplasmin (μPli)to characterize the biophysical effects of μPli on fresh, post-mortemporcine vitreous in vitro and in-situ using the non-invasive techniqueof dynamic light scattering (DLS). DLS provides information about thedynamics of particles and macromolecules in solutions and suspensions bymeasuring time fluctuations in the intensity of the scattered light.

In a DLS experiment, a constantly fluctuating speckle pattern is seen inthe far field when light passes through an ensemble of small particlessuspended in a fluid (see, Chu B., Laser light scattering: Basicprinciples and practice, Academic Press, New York, 1991). This specklepattern is the result of interference in the light paths and itfluctuates as the particles in the scattering medium perform randommovements on a time scale of 1 μsec due to the collisions betweenthemselves and the fluid molecules (Brownian motion). In the absence ofparticle-particle interactions (dilute dispersions) light scattered fromsmall particles fluctuates rapidly while light scattered from largeparticles fluctuates more slowly.

The DLS apparatus built for our studies provides dynamic informationsuch as diffusion coefficient, size, scattered intensity, andpolydispersity (measure of heterogeneity). In general, an increase inparticle sizes (from nanometers to a few microns) and an increase in thenumber or density of these particles result in an increase in scatteredlight intensity. Polydispersity is a measure of the number of distinctgroups of species with different size(s). In a DLS measurement up tothree groups differing in size can be identified since they diffuse atdifferent time scales (small particles move faster and larger slower).Therefore, a change in scattered light intensity and polydispersity cancomplement the particle size data. If after pharmacologic interventionvitreous particle sizes decrease and scattered intensity increases, thenthere is probably an increased number of smaller size molecular speciesin the solution, either due to breakdown of larger molecular species,and/or, as in the case of these experiments, an influx of 20 nmpolystyrene nanospheres that were previously excluded by the inherentvitreous structure. If polydispersity decreases, then the most likelyexplanation is that there is increased homogeneity in the population ofmolecular species in the sample, again indicative of the influx of 20 nmpolystyrene nanospheres that were previously excluded. The mostattractive features of DLS are that it is non-invasive and quantitative,works effectively for particle sizes ranging from a few nm to few um,requires small samples volume, and works reasonably well forpolydisperse or multiple size (up to 2-3 component) dispersions. Thedata presented herein was analyzed using the cumulant and exponentialsize distribution routines obtained from Brookhaven Instruments, NY.These schemes have been reviewed by Stock and Ray (Stock R. S, and RayW. H., J. Polym. Sci. 23:1393, 1985). As an example, FIG. 11 shows atypical DLS measurement in terms of a time autocorrelation function orTCF for the whole porcine vitreous (polydisperse system) and a solutionof polystyrene nanospheres of 20 nm diameter (monodisperse system).

Materials and Methods (a) Fabrication of DLS Apparatus for VitreousStudies

A new compact DLS fiber-optic probe (U.S. Pat. No. 5,973,779) wasfabricated for the vitreous studies described herein. It comprises of apair of 0.25 pitch Selfoc GRIN lenses, and it has a penetration depth of˜16 mm and a scattering angle of 160°. A fiber optic probe comprisingtwo monomode optical fibers and two GRIN lenses provides a compact andremote means of studying the dynamic characteristics of themacromolecules in the eye. Two monomode optical fibers, each housed in astainless steel ferrule, are mounted into a separate stainless steelhousing. An air gap is intentionally left between the fiber housing andthe lens housing in order to produce a tightly focused spot in thescattering volume. The two optical fibers in their housings are alignedand fixed into position off-axis with the micro lens. The two housingsare placed inside a third (outer) housing made of stainless steel, andthe back end of the housing is covered with a heat-shrink tubing. Thetwo free ends of the optical fibers were terminated with FC/PC-type maleconnectors for easy mating with the laser and photo-detector module.

The experimental set up's main components consist of a DLS compact fiberoptic probe (described above), a computer (Gateway PC 500S) containing adigital correlator card (BI-9000 Brookhaven Instruments NY), and a 635nm wavelength 1 mW solid-state laser (OZ Optics, Canada), and anavalanche photo diode detector (Perkin Elmer, Canada). The probe ismounted on an optical assembly connected through translational stagescontrolled manually to access and direct the probe to a desired locationin the eye.

Each temporal autocorrelation function (TCF) took 20 seconds to collect(except for the filtered cuvette studies which took 1 minute). The delaytime of 5 microseconds was kept constant for all the measurements. Priorto starting the vitreous study, the instrument was thoroughly tested forits stability, reliability, and reproducibility by using aqueousdispersions of polystyrene standards (latex nanospheres of 20 nmdiameter).

(b) Dynamic Light Scattering of Vitreous and Viscosity Issues

DLS is able to non-invasively provide objective quantification of theaverage diameter of particles suspended in a solution, in this case thevitreous. To calculate particle sizes accurately it is necessary toeither know, or assume the viscosity of the solvent. As previouslymentioned, it is not presently possible to accurately measure theviscosity of a non-Newtonian fluid, so assumptions must be made. Giventhat vitreous is approximately 99% water, it is reasonable to ascribethe viscosity of water to the vitreous. To test the validity of thisassumption, DLS measurements were made on whole vitreous gel, thesub-fraction of vitreous that did not pass through a strainer (gel), thesub-fraction of vitreous that did pass through the strainer (non-gel),and the sub-fraction of vitreous that passed through a 0.22 μm Milliporefilter (liquid vitreous). These studies were done in optical cuvettesand diffusing 20 nm polystyrene nanospheres were added as a tracer witha known diameter that is highly uniform. DLS measurements in wholevitreous and all the different sub-fractions yielded similar resultsindicating the vitreous microviscosity is indeed very close to that ofpure water. Microviscosity in vitreous refers to the viscosity oftrapped water in which the hyaluronan (HA) molecules and collagenbundles are suspended. The Brownian motion of HA molecules are muchfaster than the collagen bundles which are fairly large in size comparedto HA molecules. In the DLS spectra, HA molecular information isembedded at faster (short) delay times and the collagen at slower(longer) delay times. The derived information is shown in the particlesize distribution(s).

(c) Reagents

All solutions were prepared using BSS PLUS® (ALCON Labs, Ft. Worth,Tex.). Microplasmin was used at a concentration of 4 mg/ml stocksolution. Polystyrene nanospheres (Bangs Laboratories, Fishers, Ind.) of20 nm diameter and suspended in doubly distilled de-ionized water wereadded at a fixed ratio in all samples, assuring a uniform number ofnanospheres in all samples.

(d) Open Sky Model

Fresh, unfixed porcine eyes (n=11) underwent dissection of the anteriorsegment via a pars planar incision and sharp dissection of the lens/irisdiaphragm off the anterior vitreous. The dissection was carried as closeto the posterior lens capsule as possible, without incising this tissue.The eyes were maintained in a holder with the posterior segment below.

The specimens were treated with controls and μPli at doses of 0.08,0.125, 0.4, 0.6 and 0.8 mg at room temperature by placing the solutions(300 μl) that contained 60 μL of 20 nm polystyrene nanosphere solutiononto the anterior surface of the exposed vitreous body. DLS wasperformed at a single point along the central optical axis located 1, 2,and 4 mm below the vitreous/air interface. Every 15 minutes a DLSreading was obtained for a duration of 90 to 360 minutes.

(e) Closed Eye Model

A 30 G needle was used to inject 300 μL of experimental and controlsolutions containing polystyrene nanospheres and μPli at doses of0.0125, 0.025, 0.05, 0.125, 0.25, 0.5, 0.6, and 0.8 mg via a stabincision at the pars plana of intact porcine eyes (n=39). Eyes wereincubated in a holder that maintained the cornea above and the posteriorsegment below (mimicking the supine position) at 37 degrees Celsius foreither 30 or 120 minutes. Prior to placing the eye in thetemperature-controlled water bath (at all times avoiding any contactbetween the specimen and the water) and every 15 minutes thereafter, theeye was rotated about the optical axis for 10-15 seconds. Afterincubation, the eyes underwent excision of the anterior segment via apars planar incision. DLS was performed at several points (mean=18.6,s.d.=11.8) along the optical axis and along a horizontal axis (mean=30.8points, s.d.=18.0) at a depth of 4 mm behind the vitreous/air interface.

Results (a) Molecular Morphology of Porcine Vitreous

DLS measurements of whole (intact) vitreous obtained at multiple pointsalong the optical axis of the eye cup (anterior segment dissected away)demonstrate very similar findings at all points from 1.25 mm to 4.75 mmbelow the air-vitreous interface. These findings are also similar tothose obtained in excised whole vitreous placed into a cuvette, in theresidual vitreous retained after straining, and in the sub-fraction ofvitreous that passed through the strainer. All showed nearly identicalparticle size distributions. The group of larger particle sizes (to theright) has an average size of about 1000 nm and represents primarilycollagen. The smaller particle size distribution (to the left) primarilyrepresents hyaluronan (HA). This particle size distribution pattern wasthe same in eyes that received polystyrene nanosphere injection.

(b) Open Sky Model

FIG. 12 shows the TCF's obtained from a point 4 mm below theair/vitreous interface in 5 different porcine eyes and from a solutionof 20 nm polystyrene nanospheres, for comparison. It can be seen thatwith increasing doses of μPli there is a decrease in the slope of theTCF with disappearance of the slow component (larger molecular species)ultimately approaching the TCF of pure 20 nm nanospheres, i.e. allsmaller size molecular species.

The results of DLS measurements (n=5) at a depth of 1 mm followingtreatment with various solutions at room temperature: Placebo and 0.08mg were essentially the same throughout. With a dose of 0.125 mg therewas about a one-third reduction in the overall average particle sizeafter 210 minutes. There was about an 80% reduction in average particlesize after 60 minutes and nearly complete reduction of average particlesizes (only 20 nm polystyrene nanospheres were detected) with the 0.6 mgdose.

At a measurement depth of 2 mm behind the air/vitreous interface (n=3)there was about a one-third reduction in the overall average particlesize after 180 minutes (data not shown). Incubating the specimen at 37degrees Celsius and maintaining that temperature during DLS measurementsresulted in a 40% reduction in the overall average particle size aftertreatment with 0.5 mg μPli for 60 minutes in two separate eyes at twodifferent times. The total intensity measurements and the polydispersitymeasurements for all of these specimens supported and corroborated theparticle size determinations.

(c) Closed Eye Model

After a 30-minute incubation at 37 degrees Celsius with μPli dosesranging from 0.0125 to 0.8 mg there were significant changes along boththe optical and horizontal axes. In the optical axis there was aseven-fold diminution in the normalized average particle size at thehighest dose of 0.8 mg. The dose of 0.125 mg yielded a diminution in thenormalized average particle size of about one-third after 30 minutes.The lowest dose of 0.0125 mg did not appear to have any significanteffects. Across the entire range of doses the diminution in particlesize was inversely proportional to the dose of μPli (coefficient ofcorrelation=0.93). The data was fitted to a linear (straight-line)fitting routine. The normalized total intensity and polydispersity plotsfurther support the reliability of this data. The particle sizedistributions demonstrate a significant shift to the left in thedistribution of particle sizes with increasing doses of μPli. Thissuggests that μPli is effective in significantly weakening and breakingvarious chemical bonds and lysing vitreous macromolecular structure.Similar changes were detected along the horizontal axis.

There were also significant changes after a 2-hour incubation at 37degrees Celsius with μPli doses ranging from 0.0125 to 0.6 mg. At thehighest dose there was an 87.5% decrease in the normalized averageparticle size measured along the optical axis. Normalized scatteringintensity and normalized polydispersity plots corroborate this data. Theparticle size distributions showed a shift to the left with increasingdoses, attaining significant proportions with the higher doses. DLSmeasurements along a horizontal axis at a depth of 4 mm showed similarresults, with about an 85% diminution in the normalized average particlesizes and supportive findings on normalized scattering intensity andpolydispersity plots.

Comparison of the results at 30 minutes and at 2 hours demonstrates amore extensive degree of particle size breakdown with longer incubation.Consider that at a dose of 0.6 mg the normalized average particlediameter was about 20% in the 30-minute incubation and about 10% in the2-hour incubation. Thus, there was about a two-fold greater decrease inparticle size with longer incubation.

The primary parameter that derives from the DLS measurements is thediffusion coefficient. A change in the diffusion coefficient indicates achange in the vitreous macromolecular structure in response to μPlitreatment. With increasing μPli doses there is an increase in thediffusion coefficient of the vitreous. Across the range of μPli doses,diffusion coefficients were directly proportional to μPli dose. Thus,with increasing μPli dose there were decreasing diffusion coefficientsand, similar to particle size determinations, this correlation wasstatistically significant (r=0.93).

Discussion

In this experiment DLS was used to non-invasively assess molecularstructure in vitreous by measuring particle sizes, scattering intensity,and polydispersity. The results showed that there are similar DLSprofiles in various locations within whole vitreous. The most pronouncedeffects of microplasmin were upon whole vitreous incubated at 37 degreesC. for 30 min, especially at higher doses. There was a substantialdiminution in normalized average particle size and a statisticallysignificant dose-response relation was established. This suggests thatμPli would be useful as an adjunct for vitreo-retinal surgery, since a30 minute time frame is reasonable for a drug effect that does notinterfere with current surgical practices. In conjunction with the datain the previous Examples suggesting that μPli induces dehiscence at thevitreo-retinal interface, this drug appears to achieve the two desiredcomponents for pharmacologic vitreolysis: posterior vitreous detachmentand a breakdown in vitreous macromolecules with consequent increases invitreous diffusion coefficients and ultimately liquefaction.

Example 9 Microplasmin as an Adjunct to Surgical Vitrectomy

A patient presenting vitreoretinal disease in which surgical vitrectomyis indicated is treated with an injection of microplasmin prior to thesurgical vitrectomy procedure. The patient is to receive a fullophthalmic examination to establish a baseline of ocular health. Theophthalmic examination includes indirect ophthalmoscopy, slit-lampbiomicroscopy, peripheral retinal examination, intraocular pressuremeasurements, visual acuity (unaided and best corrected) symptomatology,fundus photography, fluorescein angiography, electroretinography andA-scan measurements.

Either up to 30 minutes or up to 1 day prior to the start of vitrectomy,the eye to be treated is injected with 0.025 to 0.125 mg of microplasminin 0.2 ml of the intraocular irrigating solution, BSS PLUS® or otherirrigating solution to promote the liquefaction of the vitreous and/orinduce posterior vitreous detachment.

By promoting liquefaction of the vitreous and/or posterior vitreousdetachment, the surgical vitrectomy may be made quicker and easier withless iatrogenic retinal trauma and risk of surgical complications.Allowing for more complete removal of vitreous may also lessen the riskof post-operative complications such as proliferative vitreoretinopathy.

Example 10 Treatment of Diabetic Retinopathy with Microplasmin

In this Example, a diabetic patient manifesting diabetic retinopathy istreated by the intravitreal injection of microplasmin.

The diabetic patient is to receive a full ophthalmic examination toestablish a baseline of ocular health. The ophthalmic examinationincludes indirect ophthalmoscopy, slit-lamp biomicroscopy, peripheralretinal examination, intraocular pressure measurements, visual acuity(unaided and best corrected) symptomatology, fundus photography,fluorescein angiography, electroretinography and A-scan measurements.

Following the preliminary examination, an intravitreal injection ofmicroplasmin is given to the patient's affected eye. If both eyes areaffected, they may be treated separately. The eye to be treated isinjected with a dose ranging from 0.005 mg to 0.125 mg of microplasminin 0.05 to 0.2 ml of BSS PLUS® or other irrigating solutionintravitreally to promote the liquefaction of the vitreous.

After treatment, the patients' eyes are to be examined periodically. Theextent of diabetic retinopathy presented by the patient is continuouslymonitored through periodic retinal examinations and fluoresceinangiograms to monitor the extent of venous beading, IRMA, retinalischemia, traction retinal detachment, vitreous hemorrhage, need forvitrectomy, or other complications of diabetic retinopathy.

Example 11 Effect of Microplasmin Compared to Plasmin on Speed ofFluorescein Diffusion in Post-Mortem Pig Eyes

Microplasmin is approximately one-third the molecular weight offull-length plasmin. Due to its smaller size, microplasmin is expectedto diffuse more rapidly in the vitreous than plasmin (Xu, J. et al.,cited supra). More rapid diffusion would be expected to lead to a morerapid pharmacologic effect. The present study was performed in order toconfirm both that microplasmin diffuses more rapidly than plasmin, andthat microplasmin is able to alter the vitreous gel.

Methods

Freshly isolated porcine eyes obtained from the slaughterhouse wereused. In the first experiment, one eye was injected with microplasmin(0.125 mg) and the fellow eye with vehicle control (BSS-PLUS®). Aftermaintaining both eyes at room temperature for 2 hours, both eyes werethen injected with fluorescein and incubated an additional 30 minutes.Photographs were taken at time 0, 10, 20, and 30 minutes.

In the second experiment, eyes were injected with microplasmin 0.125 mg(N=2) or plasmin 1U (supplied by Sigma, N=2), and incubated at 37degrees Celsius for 2 hours. All 4 eyes were then injected withfluorescein and incubated for an additional 30 minutes, with photographstaken at time 0, 10, 20, and 30 minutes.

Results

In the first experiment, in the control eye virtually no fluoresceindiffusion within the vitreous was observed (data not shown). However, inthe microplasmin-treated eye clear fluorescein diffusion was observed(data not shown).

In the second experiment, microplasmin-treated eyes had fluoresceindiffusion of 14% and 16%, respectively, over 20 minutes (FIG. 13), whilethe plasmin-treated eye had fluorescein diffusion of less than 10% (FIG.14).

Discussion

Microplasmin demonstrated a clear facilitation of fluorescein diffusioncompared to vehicle control. Furthermore, as predicted based onmolecular weight of microplasmin, this fluorescein diffusion was of agreater extent than that observed with full-length plasminadministration. These findings support the theoretical prediction thatmicroplasmin diffuses more rapidly than plasmin. These findings may haveclinical benefit, in allowing for more rapid pharmacologic effect.

1. A method of treating a vitreoretinal disease or disorder, or oftreating a complication of a vitreoretinal disease or disorder, of aneye of a subject, comprising contacting the aqueous humor in the eye ofthe subject with an effective amount of a microplasmin and with aneffective amount of a second agent, thereby treating the vitreoretinaldisease or disorder, or the complication of the vitreoretinal disease ordisorder, of the eye of the subject.
 2. The method according to claim 1wherein said aqueous humor is contacted with the second agent prior to,at the same time as, or after being contacted with said microplasmin. 3.The method according to claim 1 wherein the second agent is a proteinother than said microplasmin, a chemical, or another substance useful intreating or preventing an eye disorder or a complication of an eyedisorder.
 4. The method according to claim 1 wherein said second agentis selected from the group consisting of hyaluronidase, chondroitinaseABC, chondroitinase AC, chondroitinase B, chondroitin 4-sulfatase,chondroitin 6-sulfatase, β-glucuronidase, collagenase, dispase, RGDcontaining peptides, echistatin, falvoridin, anti-integrin antibody, P2Yreceptor antagonists, urea, hydroxyurea, thiourea, anti-angiogenicagents, VEGF inhibitors, PlGF inhibitors, and any combinations thereof.5. The method according to claim 4 wherein said VEGF inhibitor is ananti-VEGF antibody, a VEGF-aptamer, or a soluble VEGF receptor.
 6. Themethod according to claim 4 wherein said PlGF inhibitor is an anti-PlGFantibody, a PlGF-aptamer, or a soluble VEGF receptor.
 7. The methodaccording to claim 1 wherein said vitreoretinal disease or disorder isselected from the group consisting of retinal detachment, retinal tear,retinal neovascularization, vitreous hemorrhage, diabetic vitreoushemorrhage, proliferative diabetic retinopathy, non-proliferativediabetic retinopathy, macular pucker, macular exudates, fibrindeposition, retinal vein occlusion, retinal artery occlusion, subretinalhemorrhage, amblyopia, endophthalmitis, retinopathy of prematurity,glaucoma, retinitis pigmentosa, and any combination thereof.
 8. Themethod according to claim 1 wherein said vitreoretinal disease ordisorder is macular edema.
 9. The method according to claim 8 whereinsaid macular edema is cystoid macular edema.
 10. The method according toclaim 1 wherein said vitreoretinal disease or disorder is age-relatedmacular degeneration.
 11. The method according to claim 1 wherein saidvitreoretinal disease or disorder is macular hole.
 12. The methodaccording to claim 11 wherein said macular hole is selected from thegroup consisting of impending macular hole, full-thickness macular hole,idiopathic macular hole and traumatic macular hole.
 13. The methodaccording to claim 1 wherein said vitreoretinal disease or disorder isvitreomacular traction.
 14. The method according to claim 1 wherein saidvitreoretinal disease or disorder is incomplete posterior vitreousdetachment.
 15. The method according to claim 1 wherein saidvitreoretinal disease or disorder is associated with vitreouscontraction.
 16. The method according to claim 1 wherein saidmicroplasmin is selected from the group consisting of recombinantmicroplasmin, stabilized microplasmin, and stabilized, recombinantmicroplasmin.
 17. The method according to claim 16 wherein saidmicroplasmin is stabilized by contacting with a stabilizing agent. 18.The method according to claim 17 wherein said stabilizing agent isselected from the group consisting of tranexamic acid, hexanoic acid,lysine, serine, threonine, methionine, glutamine, alanine, glycine,isoleucine, valine, alanine aspartic acid, polyhydric alcohol, apharmaceutically acceptable carbohydrate, glucosamine, thiamine,niacinamide, an acidic buffer, a salt, and any combination thereof. 19.The method according to claim 18 wherein said acidic buffer iscomprising acetic acid, benzoic acid, carboxylic acid, citric acid,hydrochloric acid, lactic acid, malic acid or tartaric acid.
 20. Themethod according to claim 18 wherein said salt is calcium chloride,magnesium chloride, potassium chloride or sodium chloride.
 21. Themethod according to claim 16 wherein said microplasmin is purified inthe presence of a stabilizing agent.
 22. The method according to claim21 wherein said stabilizing agent is selected from the group consistingof tranexamic acid, hexanoic acid, lysine, serine, threonine,methionine, glutamine, alanine, glycine, isoleucine, valine, alanineaspartic acid, polyhydric alcohol, a pharmaceutically acceptablecarbohydrate, glucosamine, thiamine, niacinamide, an acidic buffer, asalt, and any combination thereof.
 23. The method according to claim 22wherein said acidic buffer is comprising acetic acid, benzoic acid,carboxylic acid, citric acid, hydrochloric acid, lactic acid, malic acidor tartaric acid.
 24. The method according to claim 22 wherein said saltis calcium chloride, magnesium chloride, potassium chloride or sodiumchloride.
 25. The method according to claim 1 wherein the step ofcontacting the aqueous humor with the microplasmin comprises injectingthe microplasmin into the vitreous or the aqueous humor.
 26. The methodaccording to claim 25 wherein the microplasmin is a human microplasmin.27. The method according to claim 1 wherein the subject is a human. 28.The method according to claim 1 wherein the method is performed in theabsence of mechanical vitrectomy.
 29. The method according to claim 1wherein the method is performed as an adjunct to mechanical vitrectomy.30. The method according to claim 1 wherein the effective amount of themicroplasmin is in the range of 0.005 mg to 0.2 mg per eye.
 31. Themethod according to claim 1 wherein the microplasmin consists of adouble chain polypeptide of amino acids 543 to 791 of SEQ ID NO:10wherein the peptide bond between Arg561 and Val 562 is cleaved by aplasminogen activator.
 32. The method according to claim 1 wherein themicroplasmin is an activated microplasminogen wherein saidmicroplasminogen is encoded by SEQ ID NO:3.
 33. The method according toclaim 1 wherein the microplasmin is produced by recombinant expressionin a yeast.
 34. The method according to claim 33 wherein said yeast isPichia pastoris.